Crystalline anti-human il-12 antibodies

ABSTRACT

The invention relates to batch crystallization methods for crystallizing an anti-hIL-12 antibody that allows the production of the antibody on an industrial scale, antibody crystals obtained according to the methods, compositions containing the crystals, and methods of using the crystals and the compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/920,608, filed on Mar. 29, 2007, which is incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a batch crystallization method forcrystallizing an antibody, which allows the production of the antibodyon an industrial scale; crystals of antibodies, in particular asobtained according to the disclosed method; and compositions containingthe crystals as well as methods of use of the crystals and compositions.

BACKGROUND OF THE INVENTION a) Antibody Crystals

With over 100 monoclonal antibodies (mAbs) currently being evaluated inclinical study phases 2 or 3, the mAb market is considered one of themost promising biopharmaceutical markets. Since these drugs aredelivered in single doses often exceeding 100 mg, there is an urgentneed to find suitable formulation strategies that satisfy stability,safety, and patient compliance. However, highly concentrated liquid mAbformulations show increased viscosity, hindering syringeability throughpatient friendly thin needles. Furthermore, the tendency for mAbmolecules to aggregate at such high concentrations exponentiallyincreases when compared to moderately concentrated solutions. This isunacceptable, with regard to safety and stability requirements.

Thus, the delivery of high mAb doses is reserved for large volumes,which generally have to be delivered via infusion. This way of dosing iscost intensive and significantly reduces the patient's compliance.

Therefore, pharmaceutically applicable low volume mAb crystalsuspensions for subcutaneous injection would be highly desirable.Theoretically, degradation pathways influencing the mAb integrity shouldbe significantly decelerated due to the rigidity of a crystal lattice,where motions in the protein structure are hindered. Moreover, anincrease in viscosity would be significantly reduced when comparinghighly concentrated crystal suspensions with liquid formulations. Withrespect to sustained release, it might be possible to generate or alterprotein crystals such that they dissolve slowly when brought into apatient's body. This would be a very elegant way to deliver a sustainedrelease formulation, as the extensive use of excipients and processesharming the mAb structure would be prevented.

Despite the great potential in using protein crystals as a drugsubstance, few attempts have been made to systematically evaluate thisstrategy.

A well-known exemption is insulin, which was successfully crystallizeddecades ago. Today, the use of crystal suspensions of insulin is welldescribed, offering stable and long acting formulations being wellestablished on the market. The discrepancy between the development ofinsulin crystals and crystallization of all other proteins might berelated to the fact that ordered insulin aggregates are natively formedin the pancreas. Thus, insulin crystals are easily obtained when insulinis brought in contact with an excess of zinc ions. Most other proteinstend to form unordered precipitates rather than crystals, and therefore,finding crystallization conditions for a protein is a time consuming,non-trivial task.

Despite a great interest in harvesting protein crystals for x-raydiffraction analysis, finding suitable crystallization conditions stillis an empirical science, as in principle any protein behavesdifferently. To date, no general rule has been found that might reliablypredict a successful crystallization condition for a protein of choice.Thus, obtaining crystals of a given protein always is referred to as the“bottle neck” of whatever intended application is planned later on.

Antibodies are especially hard to crystallize, due to the flexibility ofthe molecule. Nevertheless, examples of immunoglobulin crystals havebeen known for a long time. The first example of immunoglobulin crystalswere described 150 years ago by an English physician, Henry Bence Jones;he isolated crystals of an abnormal Ig light chain dimer from the urineof a myeloma patient (Jones, H. B. (1848) Philosophical Transactions ofthe Royal Society, London 138: 55-62). Such abnormal Igs have been knownever since as Bence Jones proteins. In 1938, the spontaneouscrystallization of a distinct abnormal Ig from the serum of a myelomapatient was described (von Bonsdorf, B. et al. (1938) Folia Haematologia59: 184-208), apparently an Ig heavy chain oligomer (MW 200 kDa).

Crystalline human immunoglobulins of normal structure (two heavy chainslinked to two light chains) were described over the next thirty years,again mostly isolated from myeloma patients (Putnam, F. W. (1955)Science 122: 275-7). Davies and co-workers were the first tocharacterize the structure of an intact human myeloma antibody, named“Dob”, using x-ray crystallography (Terry, W. D. et al. (1968) Nature220(164): 239-41), and they determined its three-dimensional structurein 1971 (Sarma, V. R. et al. (1971) J. Biol. Chem. 246(11): 3753-9).Their pioneering work was followed by that of others, yielding thecrystal structures of the IgG “Kol” (Huber, R. et al. (1976) Nature264(5585): 415-20), the IgG “Mcg” (Rajan, S. S. et al. (1983) Mol.Immunol. 20(7): 787-99), and a canine lymphoma IgG2a (Harris, L. J. etal. (1992 Nature 360(6402): 369-72).

Crystals of immunoglobulins retain their distinctive immunologicalactivities upon re-dissolution. Nisonoff et al. reported in 1968 on arabbit anti-p-azobenzoate antibody, “X4”, that was easily crystallized(Nisonoff, A. et al. (1968) Cold Spring Harbor Symposia on QuantitativeBiology 32: 89-93). Antibody X4 was extensively characterized beforecrystallization as well as after re-dissolution of the crystals.[¹²⁵I]-p-iodobenzoate was found to bind specifically and potently tore-dissolved X4; the redissolved crystals also exhibited multiplespecific Ouchterlony immunodiffusion reactions typical of the unpurifiedrabbit serum (Nisonoff et al., 1968). Connell and co-workers described ahuman myeloma gamma-immunoglobulin-1 kappa (IgG-κ), called “Tem”, thatcrystallized spontaneously from serum at cold temperatures (Connell, G.E. et al. (1973) Canad. J. Biochem. 51(8): 1137-41). Tem crystals werefound to be well-formed and possessed rhombohedral symmetry.Tem-containing serum was extensively characterized by agaroseimmunodiffusion techniques. Electrophoresis and immunodiffusion of are-dissolved solution of the Tem crystals showed them to be identicalwith the material obtained from the serum by cryoprecipitation, and withthe isolated myeloma protein (Connell et al., 1973).

Mills and co-workers reported in 1983 an unusualcrystallocryoglobulinemia resulting from human monoclonal antibodies toalbumin (Mills, L. E. et al. (1983) Annals of Internal Med 99(5):601-4). Here, very similar cuboidal crystals were isolated from twopatients. Redissolution of the crystals followed by electrophoresis andimmunoelectrophoresis indicated that the crystals were composed of twoprotein components, a monoclonal IgG-lambda and human serum albumin in a1:2 ratio (Jentoft, J. E. et al. (1982) Biochem. 21(2): 289-294). Thecomponents were separated on preparative scale by dissolution of theoriginal crystals followed by column chromatography. Although neitherseparated component crystallized on its own, upon recombination theoriginal bipartite complex reformed and then crystallized. Further studyof the distinctive sedimentation characteristics and immunologicalreactivity of the redissolved, separated IgG and its Fab fragment withhuman serum albumin indicated that reassociation of the two redissolved,separated components was immunologic in nature, i.e., that thecrystalline antibody once redissolved still possessed its native, highlyspecific (for human serum albumin) binding characteristics (Mills et al.1983).

Recently, Margolin and co-workers reported on the potential therapeuticuses of crystalline antibodies (Yang, M. X. et al. (2003) Proc. Natl.Acad. Sci. 100(12): 6934-6939). They found that the therapeuticmonoclonal antibody trastuzumab (Herceptin®) could be crystallized(Shenoy, B. et al. (2002) PCT Int. Appl. WO/2002/072636, (AltusBiologics Inc., USA). 173 pp.). Crystalline trastuzumab suspensions weretherapeutically efficacious in a mouse tumor model, thus demonstratingretention of biological activity by crystalline trastuzumab (Yang etal., 2003).

b) Crystallization Techniques

The crystallization of diverse proteins cannot be carried outsuccessfully using defined methods or algorithms. Certainly, there havebeen great technical advances in the last 20-30 years, as noted by theworld-renowned expert in protein crystallization, A. McPherson.McPherson provides extensive details on tactics, strategies, reagents,and devices for the crystallization of macromolecules. (McPherson, A.(1999) Crystallization of Biological Macromolecules. Cold Spring Harbor,N.Y., Cold Spring Harbor Laboratory Press, p. 159). He does not,however, provide a method to ensure that any given macromolecule canindeed be crystallized by a skilled person with reasonable expectationof success. McPherson states for example: “Whatever the procedure, noeffort must be spared in refining and optimizing the parameters of thesystem, both solvent and solute, to encourage and promote specificbonding interactions between molecules and to stabilize them once theyhave formed. This latter aspect of the problem generally depends on thespecific chemical and physical properties of the particular protein ornucleic acid being crystallized.”.

It is widely accepted by those skilled in the art of proteincrystallization that no algorithm exists to take a new protein ofinterest, apply definite process steps, and thereby obtain the desiredcrystals.

Several screening systems are commercially available (for exampleHampton 1 and 2, Wizzard I and II) which allow, on a microliter scale,to screen for potentially suitable crystallization conditions for aspecific protein. However, positive results obtained in such a screeningsystem do not necessarily allow successful crystallization in a larger,industrially applicable batch scale. Conversion of microliter-sizecrystallization trials into industrial dimensions is described to be achallenging task (see Jen, A., Merkle, H. P. (2001) Pharm. Res. 18, 11,1483).

Baldock et al. reported on a comparison of microbatch and vapordiffusion for initial screening of crystallization conditions (Baldock,P. et al. (1996) J. Crystal Growth 168(1-4):170-174. Six commerciallyavailable proteins were screened using a set of crystallizationsolutions. The screens were performed using the most common vapordiffusion method and three variants of a microbatch crystallizationmethod, including a novel evaporation technique. Out of 58crystallization conditions identified, 43 (74%) were identified bymicrobatch, while 41 (71%) were identified by vapor diffusion.Twenty-six conditions were found by both methods, and 17 (29%) wouldhave been missed if microbatch had not been used at all. This shows thatthe vapor diffusion technique, which is most commonly used in initialcrystallization screens does not guarantee positive results.

c) Anti-Human IL-12 Antibody Crystals

Human IL-12 plays a critical role in the pathology associated withseveral diseases involving immune and inflammatory responses, forexample multiple sclerosis, Crohn's disease and psoriasis. There is,therefore, a great need for suitable methods of treating such humanIL-12 related disorders. One promising therapeutic approach comprisesthe administration of pharmaceutically effective doses of anti-humanIL-12 antibodies.

Due to the role of human IL-12 in a variety of human disorders,therapeutic strategies have been designed to inhibit or counteract IL-12activity. In particular, antibodies that bind to, and neutralize, IL-12have been sought as a means to inhibit IL-12 activity. Some of theearliest antibodies were murine monoclonal antibodies (mAbs), secretedby hybridomas prepared from lymphocytes of mice immunized with IL-12(see, e.g., WO 97/15327). These murine IL-12 antibodies are, however,limited for their use in vivo due to problems associated withadministration of mouse antibodies to humans, such as short serum halflife, an inability to trigger certain human effector functions andelicitation of an unwanted immune response against the mouse antibody ina human (the “human anti-mouse antibody” (HAMA) reaction).

In general, attempts to overcome the problems associated with the use offully-murine antibodies in humans, have involved genetically engineeringthe antibodies to be more “human-like.” For example, chimericantibodies, in which the variable regions of the antibody chains aremurine-derived and the constant regions of the antibody chains arehuman-derived, have been prepared. However, because these chimeric andhumanized antibodies still retain some murine sequences, they still mayelicit an unwanted immune reaction, the human anti-chimeric antibody(HACA) reaction, especially when administered for prolonged periods.

U.S. Pat. No. 6,914,128 discloses human antibodies, preferablyrecombinant human antibodies, that specifically bind to humaninterleukin-12 (hIL-12). Preferred antibodies disclosed therein, havehigh affinity for hIL-12 and neutralize hIL-12 activity in vitro and invivo. The antibodies, or antibody portions, are useful for detectinghIL-12 and for inhibiting hIL-12 activity, e.g., in a human subjectsuffering from a disorder in which hIL-12 activity is detrimental.Nucleic acids, vectors and host cells for expressing the recombinanthuman antibodies of the invention, and methods of synthesizing therecombinant human antibodies, are also enclosed. Crystalline forms ofthe anti-hIL-12 antibodies or methods for preparing the same are notspecifically described in the '128 patent.

The problem to be solved according to the present invention is,therefore, to develop suitable crystallization conditions, in particularbatch crystallization conditions, for anti-IL-12 antibodies, and toestablish crystallization process conditions applicable to volumesrelevant for industrial antibody crystal production. At the same time, acrystallization process is established that does not make use of toxicagents, which might negatively affect the pharmaceutical applicabilityof such antibodies.

SUMMARY OF THE INVENTION

The above-mentioned problem was, surprisingly, solved by the findingthat it is possible to obtain crystals of a whole anti-human IL-12antibody in batch crystallization volumes above the microliter scale byapplying physiologically acceptable polyalkylene polyols as thecrystallization-inducing agent.

In a first aspect, the invention provides a batch crystallization methodfor crystallizing an anti-human IL-12 antibody, comprising the steps of:

-   (a) providing an aqueous solution of the IL-12 antibody in admixture    with at least one crystallization agent of the polyalkylene polyol    type, as defined in more detail below, for example polyalkylene    glycol; for example by mixing an aqueous solution of the antibody,    wherein the antibody preferably is present in dissolved form, with    an aqueous crystallization solution comprising at least one    polyalkylene glycol as crystallization agent in dissolved form, or    alternatively by adding the crystallization agent in solid form;-   (b) and incubating the aqueous crystallization mixture until    crystals of the antibody are formed.

According to a further embodiment, the method of the present inventionmay also be performed such that the crystallization mixture obtained instep a) may be supplemented with a suitable amount of pre-existinganti-human IL-12 antibody crystals as seed crystals in order to initiateor boost the crystallization.

The crystallization method of the invention generally is performed at apH of the aqueous crystallization mixture in the range of about pH 4 toabout 6.5, in particular about 4.5 to about 6.0, about 5.0 to about 5.8or about 5.3 to about 5.7, such as, for example, 5.4, 5.5 or 5.6.

Moreover, the aqueous crystallization mixture may contain at least onebuffer. The buffer may comprise an acetate component as a maincomponent, especially an alkali metal salt thereof, for example a sodiumor a potassium salt, such as sodium acetate. The salt is adjusted byaddition of an acid, in particular acetic acid, to the required pH. In apreferred embodiment of the crystallization method, the bufferconcentration (total acetate) in the aqueous crystallization mixture isabout 0 to about 0.5 M, or about 0.02 to about 0.5 M, as for exampleabout 0.05 to about 0.3 M, or about 0.07 to about 0.2 M, or about 0.09to about 0.12 M.

A “crystallization agent of the polyalkylene polyol type” is defined inmore detail below:

A skilled reader will realize that the term has to be understood broadlyand comprises polyalkylene polyols as well as derivatives thereof.

A “polyalkylene polyol” as used according to the invention is a straightor branched chain, in particular straight chain, poly-C₂-C₆-alkylenepolyol. The polyether is formed from at least one type of apolyfunctional aliphatic alcohol carrying 2 to 6, 2 to 4 and inparticular 2 or 3, preferably vicinal, hydroxyl groups and having 2 to6, in particular 2, 3 or 4 carbon atoms, preferably forming a linearcarbon backbone. Non-limiting examples are ethylene-1,2-diol (glycol),propylene-1,2-diol, propylene-1,3-diol, and n-butylene-1,3-diol andn-butylene-1,4-diol. A particularly preferred diol is glycol.

The polyalkylene polyols of the invention may be composed of one singletype of polyol or mixtures of at least to different polyols, which maybe polymerized at random or may be present as block copolymers.

Furthermore, the term “polyalkylene polyol” also comprises derivativesof the same. Non-limiting examples are alkyl esters and ethers, inparticular monoalkyl ethers and dialkyl ethers. “Alkyl” is in particulardefined as straight or branched-chain C₁-C₆-alkyl residue, inparticular, methyl, ethyl, n- or i-propyl, n-, i-, sec.-odertert.-butyl, n- or i-pentyl; and n-hexyl.

The polyalkylene polyols, in particular the polyalkylene glycols, asused according to the invention are further characterized by a widerange of molecular weights. The molecular weight range, stated asnumber- or weight average molecular weight, typically is in the range of400 to 10,000, as for example 1,000 to 8,000, or 2,000 to 6,000 3,000 to6,000 or 3,200 to 6,000, as for example 3,350 to 6,000, 3,350 to 5000,or 3,800 to 4,200, in particular about 4,000.

Particular polyalkylene polyols are polyethylene glycols (PEGs) andpolypropylene glycols (PPGs) and corresponding random or blockcopolymers. Specific examples of suitable polyols are PEG 2,000, PEG3,000, PEG 3,350, PEG 4,000, PEG 5,000 and PEG 6,000.

In particular, the polyalkylene polyol concentration, in particular thepolyethylene glycol concentration, in the crystallization mixture is inthe range of about 5 to about 30% (w/v), as for example about 7 to about15% (w/v) or about 9 to about 16% (w/v) or about 10 to about 14% (w/v)or about 11 to about 13% (w/v). Preferably, polyethylene glycol with anaverage molecular weight of about 4,000 is used in a concentration inthe crystallization mixture of about 11 to about 13% (w/v).

In a preferred embodiment of the invention, antibody protein solutionand crystallization solution are combined in a ratio of about 1:1. Thus,molarities of the buffering agents/crystallization agents in theoriginal crystallization solution are about double as high as in thecrystallization mixture.

Typically, the crystallization method is performed in a batch volume inthe range of about 1 ml to about 20,000 l, or 1 ml to about 15,000 l, or1 ml to about 12,000 l, or about 1 ml to about 10,000 l, or 1 ml toabout 6,000 l, or 1 ml to about 3,000 l, or 1 ml to about 1,000 l, or 1ml to about 100 l, as for example about 50 ml to about 8,000 ml, orabout 100 ml to about 5,000 ml, or about 1,000 ml to about 3,000 ml; orabout 1 l to about 1,000 l; or about 10 l to about 500 l.

In addition, the crystallization method of the invention may beperformed so that at least one of the following additionalcrystallization conditions is achieved:

-   a) incubation is performed for between about 1 hour to about 250    days, or 1 to 250 days or 13 to 250 days, for example about 1 to    about 30 days, or about 2 to 10 days;-   b) incubation is performed at a temperature between about 0° C. and    about 50° C., for example about 4° C. and about 37° C. or about    15° C. and about 25° C.;-   c) the antibody concentration (i.e., protein concentration) in the    crystallization mixture is in the range of about 0.5 to 280 mg/ml or    about 1 to 200 mg/ml or 1 to 100 mg/ml, for example 1.5 to 20 mg/ml,    in particular in the range of about 2 to 15 mg/ml, or 5 to 10 mg/ml.    The protein concentration may be determined according to standard    procedures for protein determination.

In a preferred embodiment, the crystallization method, for example withpolyethylene glycol as the crystallization agent, is performed such thatthe incubation is performed for between about 13 to 60 days at atemperature of about 20° C. and at an antibody concentration of about 5to 10 mg/ml.

According to a particularly preferred method, crystallization isperformed under the following conditions of the crystallization mixture:

Polyalkylene glycol: PEG 4000 10 to 15% (w/v) buffer: sodium acetate, 0to 0.3M, (total acetate) pH: 5.3 to 5.8 anti-hIL-12 concentration: 3 to10 mg/ml Temperature: 18 to 24° C. Batch volume: 1 to 100 l Agitation:None Duration: about 1 to 60 days

The crystallization mixtures as outlined above are usually obtained byadding a crystallization agent in solution or as solid to the proteinsolution. Both solutions may be, but do not have to be buffered.Crystallization agent concentration and buffer molarity in the originalcrystallization solution is usually higher than in the crystallizationmixture as it is “diluted” with the protein solution.

In a further embodiment, the crystallization method of the invention mayfurther comprise the step of drying the obtained crystals. Suitabledrying methods comprise evaporative drying, spray drying,lyophilization, vacuum drying, fluid bed drying, spray freeze drying,near critical drying, supercritical drying, and nitrogen gas drying.

In a further embodiment, the crystallization method of the invention mayfurther comprise the step of exchanging the crystallization motherliquor with a different liquid or buffered buffer, e.g., a liquid orbuffer containing a polyalkylene polyol different from the one used forcrystallization with a molar mass in the range of about 300 to 8,000Daltons or mixtures of such polyols, for example by centrifugation,diafiltration, ultrafiltration or other commonly used buffer exchangetechniques. The different liquid or buffer may also be designated as an“artificial mother liquor” which differs from the “natural”crystallization mother liquor of the crystals and prevents a dissolutionof the crystals formed.

The present invention also relates to a crystal of an anti-hIL-12antibody, obtainable by a crystallization method as defined above and ingeneral to crystals of an anti-hIL-12 antibody.

The crystals of the invention may be of different shape. The shapegenerally is designated as “sword-like”. In particular, the term alsocomprises “platelets”, “needles” or “needle-clusters” (sea urchin-like).For example, the crystals of the invention may be characterized by aneedle-like morphology with a maximum length (l) of about 2-500 μm orabout 100-300 μm and a length/diameter (l/d) ratio of about 1 to 100.The height of such needle-like crystals is roughly in the dimension ofthe diameter.

Platelets of the invention may have the following dimensions: A maximumlength (l) of about 2-500 μm or about 100-300 μm and a length/diameter(l/d) ratio of about 1 to 100. The height of such platelets isconsiderably smaller than the diameter.

Needle-clusters of the invention may have the following dimensions. Amaximum length l of about 2-200 μm or about 10-100 μm and alength/diameter (l/d) ratio of about 1 to 3.

The crystal may be obtained from a polyclonal antibody or, preferably, amonoclonal antibody.

In particular, the antibody is selected from the group consisting ofnon-chimeric or chimeric antibodies, humanized antibodies,non-glycosylated antibodies, human antibodies and mouse antibodies. Inparticular the antibody to be crystallized is a non-chimeric, humanantibody optionally further processed for improving the antigen-bindingand/or efficacy.

Preferably, the crystals are obtained from an IgG antibody such as, forexample, an IgG1, IgG2, IgG3 or IgG4 antibody. In particular, theantibody is a whole anti-human IL-12 antibody of the group IgG1.

In a preferred embodiment, the crystals are prepared from an isolatedhuman antibody, that dissociates from hIL-12 with a Kd of 1×10⁻¹⁰ M orless and a k_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance.

In particular, the crystals may be prepared from an isolated humanantibody with a light chain variable region (LCVR) comprising the aminoacid sequence of SEQ ID NO: 2 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 1.

Preferred human antibodies are, for example described in U.S. Pat. No.6,914,128.

Most preferred are crystals prepared from the antibody ABT-874.

In a further embodiment, the invention relates to a solid, liquid orsemi-solid pharmaceutical composition comprising: (a) crystals of ananti-hIL-12 antibody as defined above, and (b) at least onepharmaceutically acceptable excipient stably maintaining the antibodycrystals.

Another aspect of this invention relates to a solid, liquid orsemi-solid pharmaceutical composition comprising: (a) crystals of ananti-hIL-12 antibody as defined herein, and (b) at least onepharmaceutically acceptable excipient encapsulating or embedding theantibody crystals. The composition may further comprise (c) at least onepharmaceutically acceptable excipient stably maintaining the antibodycrystals. Moreover, encapsulation and embedding may be implemented inconjunction.

In particular, the compositions of the invention may have an antibodycrystal concentration higher than about 1 mg/ml, in particular about 200mg/ml or more, for example about 200 to about 600 mg/ml, or about 300 toabout 500 mg/ml.

The excipients may comprise at least one polymeric, optionallybiodegradable carrier or at least one oil or lipid carrier.

The polymeric carrier may be one or more polymer selected from the groupconsisting of: poly (acrylic acid), poly (cyanoacrylates), poly (aminoacids), poly (anhydrides), poly (depsipeptide), poly (esters), poly(lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly(ethylene glycol), poly (hydroxypropyl) methacrylamide, poly (organo)phosphazene, poly (ortho esters), poly (vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends andcopolymers thereof.

The oil (or oily liquid) may be one or more oil (or oily liquid)selected from the group consisting of oleaginous almond oil, corn oil,cottonseed oil, ethyl oleate, isopropyl myristate, isopropyl palmitate,mineral oil, light mineral oil, octyldodecanol, olive oil, peanut oil,persic oil, sesame oil, soybean oil, squalane, liquid triglycerides,liquid waxes, and higher alcohols.

The lipid carrier may be one or more lipid selected from the groupconsisting of fatty acids and salts of fatty acids, fatty alcohols,fatty amines, mono-, di-, and triglycerides of fatty acids,phospholipids, glycolipids, sterols and waxes and related similarsubstances. Waxes are further classified in natural and syntheticproducts. Natural materials include waxes obtained from vegetable,animal or minerals sources such as beeswax, carnauba or montanwax.Chlorinated naphthalenes and ethylenic polymers are examples forsynthetic wax products.

In a preferred embodiment, the composition is an injectable compositioncomprising anti-hIL-12 antibody crystals as defined above and having anantibody crystal concentration in the range of about 10 to about 400mg/ml or about 50 to about 300 mg/ml.

In a further aspect the invention relates to a crystal slurry comprisinganti-hIL-12 antibody crystals as defined above having an antibodycrystal concentration higher than about 100 mg/ml, for example about 150to about 600 mg/ml, or about 200 to about 400 mg/ml.

The present invention also relates to a method for treating a mammalcomprising the step of administering to the mammal an effective amountof whole anti-hIL-12 antibody crystals as defined above or an effectiveamount of the composition as defined above. Preferably, the compositionis administered by parenteral route, oral route, or by injection.

Furthermore, the present invention relates to a method of treating ahIL-12-related disorder in a subject that comprises administering atherapeutically effective amount of antibody crystals as defined above.

In particular, the hIL-12-related disorder is selected from:

rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lymearthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,systemic lupus erythematosus, Crohn's disease, ulcerative colitis,inflammatory bowel disease, insulin dependent diabetes mellitus,thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, atopic dermatitis, graft versus host disease, organtransplant rejection, acute or chronic immune disease associated withorgan transplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadic,polyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia areata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis C, common variedimmunodeficiency (common variable hypogammaglobulinaemia), dilatedcardiomyopathy, female infertility, ovarian failure, premature ovarianfailure, fibrotic lung disease, cryptogenic fibrosing alveolitis,post-inflammatory interstitial lung disease, interstitial pneumonitis,connective tissue disease associated interstitial lung disease, mixedconnective tissue disease associated lung disease, systemic sclerosisassociated interstitial lung disease, rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus associated lungdisease, dermatomyositis/polymyositis associated lung disease,Sjodgren's disease associated lung disease, ankylosing spondylitisassociated lung disease, vasculitic diffuse lung disease, haemosiderosisassociated lung disease, drug-induced interstitial lung disease,radiation fibrosis, bronchiolitis obliterans, chronic eosinophilicpneumonia, lymphocytic infiltrative lung disease, postinfectiousinterstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediatedhypoglycemia, type B insulin resistance with acanthosis nigricans,hypoparathyroidism, acute immune disease associated with organtransplantation, chronic immune disease associated with organtransplantation, osteoarthrosis, primary sclerosing cholangitis,idiopathic leucopenia, autoimmune neutropenia, renal disease NOS,glomerulonephritides, microscopic vasulitis of the kidneys, lymedisease, discoid lupus erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes),insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia,idiopathic thrombocytopenia, autoimmune thyroid disease,hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto'sdisease), atrophic autoimmune hypothyroidism, primary myxoedema,phacogenic uveitis, primary vasculitis and vitiligo. The humanantibodies, and antibody portions of the invention can be used to treatautoimmune diseases, in particular those associated with inflammation,including, rheumatoid spondylitis, allergy, autoimmune diabetes,autoimmune uveitis.

Moreover, the present invention relates to the use of whole anti-hIL-12antibody crystals as defined above for preparing a pharmaceuticalcomposition for treating a hIL-12-related disease as defined above.

Finally, the present invention provides anti-hIL-12 antibody crystals asdefined above for use in medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the presentinvention, as well as the invention itself, will be more fullyunderstood from the following description of preferred embodiments whenread together with the accompanying drawings, in which:

FIG. 1 shows a light micrograph of ABT-874 crystals in crystallization.

FIGS. 2 to 5 show SEMs of ABT-874 crystals at different magnification;FIG. 2: 1,250×; FIG. 3: 10,000×; FIG. 4: 3,227×; FIG. 5: 15,000×.

FIGS. 6A-C show the results of Capillary Isoelectric Focusing (cIEF)Experiments with ABT-874; FIG. 6A) ABT-874 crystal buffer and pl markersof pl 8.4, 8.5, 10.1 and 10.4; FIG. 6B) ABT-874 crystals; same pl markerand characteristic ABT-874 signal at pl=9,29; FIG. 6C) ReferenceStandard; same pl marker and characteristic ABT-874 signal at pl=9,29.

FIG. 7 shows light microscopic pictures of crystals (needle-clusters)obtained according to Example 28 (crystallization with agitation).

FIG. 8 shows light microscopic pictures of crystals (needles) obtainedaccording to Example 32 (crystallization without agitation).

FIG. 9 shows light microscopic pictures of crystals (needles) obtainedaccording to Example 33 (crystallization without agitation).

FIG. 10 shows light microscopic pictures of crystals (needles) obtainedaccording to Example 34b (crystallization without agitation).

FIGS. 11A-B show second derivative IR spectra of ABT-874 samples. FIG.11A shows spectra of crystal suspension recorded with an BioATR cell.FIG. 11B shows spectra of redissolved crystals recorded with an AquaSpeccell. Solid lines represent samples from crystalline ABT-874, dashedlines represent liquid standards. An offset between sample and standardwas inserted for better illustration.

FIGS. 12A-B show second derivative IR spectra of ABT-874 samples, 50mg/mL crystalline protein in 22% PEG 4,000 buffer in 0.1 M sodiumacetate buffer, pH 5.5, stored for 3 months at 25° C. FIG. 12A showsspectra of crystal suspension recorded with an BioATR cell. FIG. 12Bshows spectra of redissolved crystals recorded with an AquaSpec cell. Anoffset between sample and standard was inserted for better illustration.

FIG. 13: 40 mL batch crystallization of ABT-874 with and without seeding(e.g., using 3.25% crystallized protein as seeding material in relationto ABT-874 mass from the batch). R² are 0.9711 for non seeded, and0.9763 for the seeded batch, respectively.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

A “batch method of crystallization” comprises the step of adding thecrystallization solution comprising the crystallization agent,preferably in dissolved form, to the solution of the antibody to becrystallized.

A “micro scale crystallization method”, which may for example be basedupon vapor diffusion, comprises the steps of mixing a small volume ofantibody solution in the microliter range with a reservoir buffercontaining a crystallization agent; placing a droplet of the mixture ina sealed container adjacent to an aliquot of the reservoir buffer;allowing exchange of solvent between the droplet and the reservoir byvapor diffusion, during which the solvent content in the droplet changesand crystallization may be observed if suitable crystallizationconditions are reached.

A “crystallization agent”, e.g., a polyethylene glycol, favors crystalformation of the antibody to be crystallized.

A “crystallization solution” contains a crystallization agent indissolved form. Preferably the solution is an aqueous system, i.e., theliquid constituents thereof predominantly consist of water. For example,80 to 100 wt.-% or 95 to 100 wt.-% or 98 to 100 wt.-% may be water.

Antibody “crystals” are one form of the solid state of matter of theprotein, which is distinct from a second solid form, i.e., the amorphousstate, which exists essentially as an unorganized, heterogeneous solid.Crystals have a regular three-dimensional structure, typically referredto as a lattice. An antibody crystal comprises a regularthree-dimensional array of antibody molecules (see Giege, R. andDucruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, aPractical Approach, 2nd ed., pp. 1-16, Oxford University Press, New York(1999)).

A “whole” or “intact” anti-hIL-12 antibody as crystallized according tothis invention, is a functional antibody that is able to recognize andbind to its antigen human IL-12 in vitro and/or in vivo. The antibodymay initiate subsequent immune system reactions of a patient associatedwith antibody-binding to its antigen, in particular Direct Cytotoxicity,Complement-Dependent Cytotoxicity (CDC), and Antibody-DependentCytotoxicity (ADCC). The antibody molecule has a structure composed oftwo identical heavy chains (MW each about 50 kDa) covalently bound toeach other, and two identical light chains (MW each about 25 kDa), eachcovalently bound to one of the heavy chains. The four chains arearranged in a classic “Y” motif. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The complete antibody molecule has two antigen binding sites,i.e., is “bivalent”. The two antigen binding sites are specific for onehIL-12 antigen, i.e., the antibody is “mono-specific”.

“Monoclonal antibodies” are antibodies that are derived from a singleclone of B lymphocytes (B cells), and recognize the same antigenicdeterminant. Whole monoclonal antibodies are those that have theabove-mentioned classic molecular structure that includes two completeheavy chains and two complete light chains. Monoclonal antibodies areroutinely produced by fusing the antibody-producing B cell with animmortal myeloma cell to generate B cell hybridomas, which continuallyproduce monoclonal antibodies in cell culture. Other production methodsare available, for example, expression of monoclonal antibodies inbacterial, yeast, insect, or mammalian cell culture using phage-displaytechnology; in vivo production in genetically modified animals, such ascows, goats, pigs, rabbits, chickens, or in transgenic mice which havebeen modified to contain and express the entire human B cell genome; orproduction in genetically modified plants, such as tobacco and corn.Anti-hIL-12 antibodies from all such sources may be crystallizedaccording to this invention.

The monoclonal antibodies to be crystallized according to the inventioninclude “chimeric” anti-hIL-12 antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass. For example, amouse/human chimera contains the variable antigen-binding portions of amurine antibody and the constant portions derived from a human antibody.

“Humanized” forms of non-human (e.g., murine) anti-hIL-12 antibodies arealso encompassed by the invention. Humanized antibodies are chimericantibodies that contain minimal sequence derived from a non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins in which residues from one or more complementaritydetermining regions (CDRs) or hypervariable loops (HVLs) of the humanimmunoglobulin are replaced by residues from a CDR or HVL of a non-humanspecies, such as mouse, rat, rabbit or nonhuman primate, having thedesired functionality. Framework region (FR) residues of the humanimmunoglobulin may replaced by corresponding non-human residues toimprove antigen binding affinity. Furthermore, humanized antibodies maycomprise residues that are found neither in the corresponding human ornon-human antibody portions. These modifications may be necessary tofurther improve antibody efficacy.

A “human antibody” or “fully human antibody” is one, which has an aminoacid sequence which corresponds to that of an antibody produced by ahuman or which is recombinantly produced. The term “human antibody”, asused herein, is intended to include antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library,antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see, e.g., Taylor, L. D. et al. (1992)Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hIL-12 activity”), is intended to refer to an antibody whosebinding to hIL-12 results in inhibition of the biological activity ofhIL-12. This inhibition of the biological activity of hIL-12 can beassessed in vitro or in vivo by measuring one or more indicators ofhIL-12 biological activity, such as hIL-12-induced cell proliferationand hIL-12 binding to hIL-12 receptors or hIL-12 induced decrease ofwhite blood cells in vivo.

These indicators of hIL-12 biological activity can be assessed by one ormore of several standard in vitro or in vivo assays known in the art.Preferably, the ability of an antibody to neutralize hIL-12 activity isassessed by inhibition of hIL-12-induced cell proliferation inphytohemagglutinin blasts and murine 2D6 cells.

An “affinity matured” anti-hIL-12 antibody is one with one or morealterations in one or more hypervariable regions, which result in animprovement in the affinity of the antibody for antigen, compared to aparent antibody. Affinity matured antibodies will have nanomolar or evenpicomolar affinities values for the target antigen. Affinity maturedantibodies are produced by procedures known in the art. Marks et al.(1992) Bio/Technology 10:779-783 describes affinity maturation by VH andVL domain shuffling. Random mutagenesis of CDR and/or framework residuesis described by Barbas et al. (1994) Proc. Nat. Acad. Sci. USA91:3809-3813 (1994); Scier et al. (1995) Gene 169:147-155; Yelton et al.(1995) J. Immunol. 155:1994-2004; Jackson et al. (1995) J. Immunol.154(7):3310-9; and Hawkins et al. (1992) J. Mol. Biol. 226:889-896.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hIL-12 is substantially free of antibodies that specifically bindantigens other than hIL-12). An isolated antibody that specificallybinds hIL-12 may, however, have cross-reactivity to other antigens, suchas hIL-12 molecules from other species. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

The phrase “human interleukin 12” (abbreviated herein as hIL-12, orIL-12), as used herein, includes a human cytokine that is secretedprimarily by macrophages and dendritic cells. The term includes aheterodimeric protein comprising a 35 kD subunit (p35) and a 40 kDsubunit (p40) which are both linked together with a disulfide bridge.The heterodimeric protein is referred to as a “p70 subunit”. Thestructure of human IL-12 is described further in, for example,Kobayashi, et al. (1989) J. Exp Med. 170:827-845; Seder, et al. (1993)Proc. Natl. Acad. Sci. 90:10188-10192; Ling, et al. (1995) J. Exp Med.154:116-127; Podlaski, et al. (1992) Arch. Biochem. Biophys.294:230-237. The term human IL-12 is intended to include recombinanthuman IL-12 (rh IL-12), which can be prepared by standard recombinantexpression methods.

The term “k_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

A “functional equivalent” of a specific “parent” anti-hIL-12 antibody ascrystallized according to the invention is one that shows the sameantigen-specificity, but differs however with respect to the molecularcomposition of the “parent” antibody on the amino acid level orglycosylation level. The differences may be merely such that thecrystallization conditions do not deviate from the parameter ranges asdisclosed herein.

“Encapsulation” of antibody crystals refers to a formulation where theincorporated crystals are individually coated by at least one layer of acoating material. In a preferred embodiment, such coated crystals mayhave a sustained dissolution rate.

“Embedding” of antibody crystals refers to a formulation where thecrystals, which might be encapsulated or not, are incorporated into asolid, liquid or semi-solid carrier in a disperse manner. Such embeddedcrystallized antibody molecules may be released or dissolved in acontrolled, sustained manner from the carrier.

B. Method of Crystallization

The crystallization method of the invention is in principle applicableto any anti-hIL-12 antibody. The antibody may be a polyclonal antibodyor, preferably, a monoclonal antibody. The antibody may be chimericantibodies, humanized antibodies, human antibodies or non-human, as forexample mouse antibodies, each in glycosylated or non-glycosylated form.In particular the method is applicable to ABT-874 and functionalequivalents thereof.

Preferably the anti-hIL-12 antibody is an IgG antibody, in particular ananti human IL-12 antibody of the group IgG1.

Unless otherwise stated the crystallization method of the inventionmakes use of technical equipment, chemicals and methodologies well knownin the art. However, as explained above, the present invention is basedon the surprising finding that the selection of specific crystallizationconditions, in particular, the selection of specific crystallizationagents, optionally further combined with specific pH conditions and/orconcentration ranges of the corresponding agents (buffer, antibody,crystallization agent), allows for the first time to preparereproducibly and in a large scale stable crystals of antibodies, inparticular non-chimeric, human antibodies, directed against hIL-12,which can be further processed to form an active ingredient of asuperior, highly advantageous pharmaceutical composition.

The starting material for performing the crystallization method normallycomprises a concentrated solution of the antibody to be crystallized.The protein concentration may, for example, be in the range of about 5to about 300 mg/ml, preferably about 5 to about 200 mg/ml, preferablyabout 5 to about 75 mg/ml. The solution may contain additivesstabilizing the dissolved antibody, and it may be advisable to removethe additives in advance. This can be achieved by performing a bufferexchange step.

Preferably the starting material for performing the crystallizationcontains the antibody in an aqueous solution, having a pH adjusted inthe range of about 3.2 to about 8.2, or about 4.0 to about 8.0, inparticular about 4.5 to about 6.5, preferably about 5.0 to about 5.5.The pH may be adjusted by means of a suitable buffer applied in a finalconcentration of about 1 to about 500 mM, in particular about 1 to about100 mM or 1 to about 10 mM. The solution may contain additives, as forexample in a proportion of about 0.01 to about 15, or about 0.1 to about5, or about 0.1 to about 2 wt.-% based on the total weight of thesolution, such as salts, sugars, sugar alcohols and surfactants, inorder to further stabilize the solution. The excipients are preferablybe selected from physiologically acceptable compounds, routinely appliedin pharmaceutical preparations. As non-limiting examples, excipientsinclude salts, such as NaCl; surfactants, such as polysorbate 80 (Tween80), polysorbate 20 (Tween 20); sugars, such as sucrose, trehalose;sugar alcohols, such as mannitol, sorbitol; and buffer agents, such asphosphate-based buffer systems, sodium and potassium hydrogen phosphatebuffers as defined above, acetate buffer, phosphate buffer, citratebuffer, TRIS buffer, maleate buffer or succinate buffer, histidinebuffer; amino acids, such as histidine, arginine and glycine.

The buffer exchange may be performed by means of routine methods, forexample dialysis, diafiltration or ultrafiltration.

The initial protein concentration of the aqueous solution used asstarting material should be in the range of about 0.5 to about 200 orabout 1 to about 50 mg/ml.

Depending on the intended final batch size (which may be in the range of1 ml to 20,000 litres) an initial volume of the aqueous antibodysolution is placed in an appropriate container (as for example a vessel,bottle or tank) made of inert material, as for example glass, polymer ormetal. The initial volume of the aqueous solution may correspond toabout 30 to 80%, normally about 50% of the final batch size.

If necessary the solution after having been filled into the containerwill be brought to standardized conditions. In particular, thetemperature will be adjusted in the range of about 4° C. and about 37°C.

Then the crystallization solution, containing the crystallization agentin an appropriate concentration, optionally pre-conditioned in the sameway as the antibody solution, is added to the antibody solution.

The addition of the crystallization solution is performed continuouslyor discontinuously optionally under gentle agitation in order tofacilitate mixing of the two liquids. Preferably the addition isperformed under conditions where the protein solution is provided underagitation and the crystallization solution (or agents in its solid from)is/are added in a controlled manner.

The formation of the antibody crystals is initiated by applying apolyalkylene polyol as defined above, in particular a polyalkyleneglycol, and preferably a polyethylene glycol (PEG), or a mixture of atleast two different polyalkylene glycols as defined above as thecrystallization agent. The crystallization solution contains the agentin a concentration, which is sufficient to afford a final concentrationof the polyalkylene polyol in the crystallization mixture in the rangeof about 5 to 30% (w/v).

Preferably, the crystallization solution additionally contains an acidicbuffer, e.g., different from that of the antibody solution, in aconcentration suitable to allow the adjustment of the pH of thecrystallization mixture in the range of about 4 to 6.

After having finished the addition of the crystallization solution, theobtained mixture may be further incubated for about 1 hour to about 250days in order to obtain a maximum yield of antibody crystals. Ifappropriate, the mixture may, for example, be agitated, gently stirred,rolled or otherwise moved.

Finally, the crystals obtained may be separated by known methods, forexample filtration or centrifugation, as for example by centrifugationat about 200-20,000 rpm, preferably 500-2,000 rpm, at room temperatureor 4° C. The remaining mother liquor may be discarded or furtherprocessed.

If necessary, the isolated crystals may be washed and subsequentlydried, or the mother liquor can be exchanged by a different solventsystem suitable for storage and/or final use of the antibodies suspendedtherein.

Antibody crystals formed according to the present invention may vary intheir shape. as already explained above For therapeutic administration,the size of the crystals will vary depending on the route ofadministration, for example, for subcutaneous administration the size ofthe crystals may be larger than for intravenous administration.

The shape of the crystals may be altered by adding specific additionaladditives to the crystallization mixture, as has been previouslydescribed for both protein crystals and crystals of low molecular weightorganic and inorganic molecules.

If necessary, it may be verified that the crystals are in fact crystalsof the antibody. Crystals of an antibody can be analyzed microscopicallyfor birefringence. In general, crystals, unless of cubic internalsymmetry, will rotate the plane of polarization of polarized light. Inyet another method, crystals can be isolated, washed, resolubilized andanalyzed by SDS-PAGE and, optionally, stained with an anti-Fc receptorantibody. Optionally, the resolubilized antibody can also be tested forbinding to its hIL-12 utilizing standard assays.

Crystals as obtained according to the invention may also be crosslinkedto one another. Such crosslinking may enhance stability of the crystals.Methods for crosslinking crystals described, for example, in U.S. Pat.No. 5,849,296. Crystals can be crosslinked using a bifunctional reagentsuch as glutaraldehyde. Once crosslinked, crystals can be lyophilizedand stored for use, for example, in diagnostic or therapeuticapplications.

In some cases, it may be desirable to dry the crystal. Crystals may bedried by means of inert gases, like nitrogen gas, vacuum oven drying,lyophilization, evaporation, tray drying, fluid bed drying, spraydrying, vacuum drying or roller drying. Suitable methods are well known.

Crystals formed according to the invention can be maintained in theoriginal crystallization solution, or they can be washed and combinedwith other substances, like inert carriers or ingredients to formcompositions or formulations comprising crystals of the invention. Suchcompositions or formulations can be used, for example, in therapeuticand diagnostic applications.

A preferred embodiment is to combine a suitable carrier or ingredientwith crystals of the invention in that way that crystals of theformulation are embedded or encapsulated by an excipient. Suitablecarriers may be taken from the non limiting group of: poly (acrylicacid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides),poly (depsipeptide), poly (esters), poly (lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly (β-hydroxybutryate), poly(caprolactone), poly (dioxanone); poly (ethylene glycol), poly(hydroxypropyl) methacrylamide, poly (organo) phosphazene, poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydridealkyl vinyl ether copolymers, pluronic polyols, albumin, alginate,cellulose and cellulose derivatives, collagen, fibrin, gelatin,hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof, SAIB, fatty acids andsalts of fatty acids, fatty alcohols, fatty amines, mono-, di-, andtriglycerides of fatty acids, phospholipids, glycolipids, sterols andwaxes and related similar substances. Waxes are further classified innatural and synthetic products. Natural materials include waxes obtainedfrom vegetable, animal or minerals sources such as beeswax, carnauba ormontanwax. Chlorinated naphthalenes and ethylenic polymers are examplesfor synthetic wax products.

C. Compositions

Another aspect of the invention relates to compositions/formulationscomprising anti-hIL-12 antibody crystals in combination with at leastone carrier/excipient.

The formulations may be solid, semisolid or liquid.

Formulations of the invention are prepared, in a form suitable forstorage and/or for use, by mixing the antibody having the necessarydegree of purity with a physiologically acceptable additive, likecarrier, excipient and/or stabilizer (see for example Remington'sPharmaceutical Sciences, 16th Edn., Osol, A. Ed. (1980)), in the form ofsuspensions, lyophilized or dried in another way. Optionally furtheractive ingredients, as for example different antibodies, biomolecules,chemically or enzymatically synthesized low-molecular weight moleculesmay be incorporated as well.

Acceptable additives are non-toxic to recipients at the dosages andconcentrations employed. Nonlimiting examples thereof include:

Acidifying agents, like acetic acid, citric acid, fumaric acid,hydrochloric acid, malic acid, nitric acid, phosphoric acid, dilutedphosphoric acid, sulfuric acid, tartaric acid.

Aerosol propellants, like butane, dichlorodifluoromethane,dichlorotetrafluoroethane, isobutane, propane,trichloromonofluoromethane.

Air displacements, like carbon dioxide, nitrogen;

Alcohol denaturants, like methyl isobutyl ketone, sucrose octacetate;

Alkalizing agents, like ammonia solution, ammonium carbonate,diethanolamine, diisopropanolamine, potassium hydroxide, sodiumbicarbonate, sodium borate, sodium carbonate, sodium hydroxide,trolamine;

Antifoaming agents, like dimethicone, simethicone.

Antimicrobial preservatives, like benzalkonium chloride, benzalkoniumchloride solution, benzelthonium chloride, benzoic acid, benzyl alcohol,butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol,cresol, dehydroacetic acid, ethylparaben, methylparaben, methylparabensodium, phenol, phenylethyl alcohol, phenylmercuric acetate,phenylmercuric nitrate, potassium benzoate, potassium sorbate,propylparaben, propylparaben sodium, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimerosal, thymol.

Antioxidants, like ascorbic acid, ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, hypophosphorous acid,monothioglycerol, propyl gallate, sodium formaldehyde sulfoxylate,sodium metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol,tocopherols excipient;

Buffering agents, like acetic acid, ammonium carbonate, ammoniumphosphate, boric acid, citric acid, lactic acid, phosphoric acid,potassium citrate, potassium metaphosphate, potassium phosphatemonobasic, sodium acetate, sodium citrate, sodium lactate solution,dibasic sodium phosphate, monobasic sodium phosphate, histidine.

Chelating agents, like edetate disodium, ethylenediaminetetraacetic acidand salts, edetic acid;

Coating agents, like sodium carboxymethylcellulose, cellulose acetate,cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceuticalglaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose,hydroxypropyl methylcellulose phthalate, methacrylic acid copolymer,methylcellulose, polyethylene glycol, polyvinyl acetate phthalate,shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax,zein, poly amino acids, other polymers like PLGA etc., and SAIB.

Coloring agents, like ferric oxide.

Complexing agents, like ethylenediaminetetraacetic acid and salts(EDTA), edetic acid, gentisic acid ethanolamide, oxyquinoline sulfate.

Desiccants, like calcium chloride, calcium sulfate, silicon dioxide.

Emulsifying and/or solubilizing agents, like acacia, cholesterol,diethanolamine (adjunct), glyceryl monostearate, lanolin alcohols,lecithin, mono- and di-glycerides, monoethanolamine (adjunct), oleicacid (adjunct), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene50 stearate, polyoxyl 35 caster oil, polyoxyl 40 hydrogenated castoroil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate80, propylene glycol diacetate, propylene glycol monostearate, sodiumlauryl sulfate, sodium stearate, sorbitan monolaurate, soritanmonooleate, sorbitan monopalmitate, sorbitan monostearate, stearic acid,trolamine, emulsifying wax.

Filtering aids, like powdered cellulose, purified siliceous earth.

Flavors and perfumes, like anethole, benzaldehyde, ethyl vanillin,menthol, methyl salicylate, monosodium glutamate, orange flower oil,peppermint, peppermint oil, peppermint spirit, rose oil, stronger rosewater, thymol, tolu balsam tincture, vanilla, vanilla tincture,vanillin.

Glidant and/or anticaking agents, like calcium silicate, magnesiumsilicate, colloidal silicon dioxide, talc.

Humectants, like glycerin, hexylene glycol, propylene glycol, sorbitol;

Ointment bases, like lanolin, anhydrous lanolin, hydrophilic ointment,white ointment, yellow ointment, polyethylene glycol ointment,petrolatum, hydrophilic petrolatum, white petrolatum, rose waterointment, squalane.

Plasticizers, like castor oil, lanolin, mineral oil, petrolatum, benzylbenzyl formate, chlorobutanol, diethyl pthalate, sorbitol, diacetylatedmonoglycerides, diethyl phthalate, glycerin, glycerol, mono- anddi-acetylated monoglycerides, polyethylene glycol, propylene glycol,triacetin, triethyl citrate, ethanol.

Polypeptides, like low molecular weight (less than about 10 residues);

Proteins, such as serum albumin, gelatin, or immunoglobulins;

Polymer membranes, like cellulose acetate membranes.

Solvents, like acetone, alcohol, diluted alcohol, amylene hydrate,benzyl benzoate, butyl alcohol, carbon tetrachloride, chloroform, cornoil, cottonseed oil, ethyl acetate, glycerin, hexylene glycol, isopropylalcohol, methyl alcohol, methylene chloride, methyl isobutyl ketone,mineral oil, peanut oil, polyethylene glycol, propylene carbonate,propylene glycol, sesame oil, water for injection, sterile water forinjection, sterile water for irrigation, purified water, liquidtriglycerides, liquid waxes, higher alcohols.

Sorbents, like powdered cellulose, charcoal, purified siliceous earth,Carbon dioxide sorbents, barium hydroxide lime, soda lime.

Stiffening agents, like hydrogenated castor oil, cetostearyl alcohol,cetyl alcohol, cetyl esters wax, hard fat, paraffin, polyethyleneexcipient, stearyl alcohol, emulsifying wax, white wax, yellow wax.

Suppository bases, like cocoa butter, hard fat, polyethylene glycol;

Suspending and/or viscosity-increasing agents, like acacia, agar,alginic acid, aluminum monostearate, bentonite, purified bentonite,magma bentonite, carbomer 934p, carboxymethylcellulose calcium,carboxymethylcellulose sodium, carboxymethylcellulose sodium 12,carrageenan, microcrystalline and carboxymethylcellulose sodiumcellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesiumaluminum silicate, methylcellulose, pectin, polyethylene oxide,polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide,colloidal silicon dioxide, sodium alginate, tragacanth, xanthan gum;

Sweetening agents, like aspartame, dextrates, dextrose, excipientdextrose, fructose, mannitol, saccharin, calcium saccharin, sodiumsaccharin, sorbitol, solution sorbitol, sucrose, compressible sugar,confectioners sugar, syrup;

Tablet binders, like acacia, alginic acid, sodiumcarboxymethylcellulose, microcrystalline cellulose, dextrin,ethylcellulose, gelatin, liquid glucose, guar gum, hydroxypropylmethylcellulose, methylcellulose, polyethylene oxide, povidone,pregelatinized starch, syrup.

Tablet and/or capsule diluents, like calcium carbonate, dibasic calciumphosphate, tribasic calcium phosphate, calcium sulfate, microcrystallinecellulose, powdered cellulose, dextrates, dextrin, dextrose excipient,fructose, kaolin, lactose, mannitol, sorbitol, starch, pregelatinizedstarch, sucrose, compressible sugar, confectioner's sugar;

Tablet disintegrants, like alginic acid, microcrystalline cellulose,croscarmellose sodium, corspovidone, polacrilin potassium, sodium starchglycolate, starch, pregelatinized starch.

Tablet and/or capsule lubricants, like calcium stearate, glycerylbehenate, magnesium stearate, light mineral oil, polyethylene glycol,sodium stearyl fumarate, stearic acid, purified stearic acid, talc,hydrogenated vegetable oil, zinc stearate;

Tonicity agent, like dextrose, glycerin, mannitol, potassium chloride,sodium chloride Vehicle: flavored and/or sweetened aromatic elixir,compound benzaldehyde elixir, iso-alcoholic elixir, peppermint water,sorbitol solution, syrup, tolu balsam syrup.

Vehicles, like oleaginous almond oil, corn oil, cottonseed oil, ethyloleate, isopropyl myristate, isopropyl palmitate, mineral oil, lightmineral oil, myristyl alcohol, octyldodecanol, olive oil, peanut oil,persic oil, sesame oil, soybean oil, squalane; solid carrier sugarspheres; sterile bacteriostatic water for injection, bacteriostaticsodium chloride injection, liquid triglycerides, liquid waxes, higheralcohols

Water repelling agents, like cyclomethicone, dimethicone, simethicone;

Wetting and/or solubilizing agents, like benzalkonium chloride,benzethonium chloride, cetylpyridinium chloride, docusate sodium,nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer, polyoxyl 35 castoroil, polyoxyl 40, hydrogenated castor oil, polyoxyl 50 stearate,polyoxyl 10 oleyl ether, polyoxyl 20, cetostearyl ether, polyoxyl 40stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate80, sodium lauryl sulfate, sorbitan monolaureate, sorbitan monooleate,sorbitan monopalmitate, sorbitan monostearate, and tyloxapol.

The crystals may be combined with a polymeric carrier to provide forstability and/or sustained release. Such polymers include biocompatibleand biodegradable polymers. A polymeric carrier may be a single polymertype or it may be composed of a mixture of polymer types. Nonlimitingexamples of polymeric carriers have already been stated above.

Examples of preferred ingredients or excipients include:

salts of amino acids such as glycine, arginine, aspartic acid, glutamicacid, lysine, asparagine, glutamine, proline, histidine;

monosaccharides, such as glucose, fructose, galactose, mannose,arabinose, xylose, ribose;

disaccharides, such as lactose, trehalose, maltose, sucrose;

polysaccharides, such as maltodextrins, dextrans, starch, glycogen;

alditols, such as mannitol, xylitol, lactitol, sorbitol;

glucuronic acid, galacturonic acid;

cyclodextrins, such as methyl cyclodextrin,hydroxypropyl-(3-cyclodextrin)

inorganic salts, such as sodium chloride, potassium chloride, magnesiumchloride, phosphates of sodium and potassium, boric acid ammoniumcarbonate and ammonium phosphate;

organic salts, such as acetates, citrate, ascorbate, lactate;

emulsifying or solubilizing agents like acacia, diethanolamine, glycerylmonostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol,poloxamer, polysorbates, sodium lauryl sulfate, stearic acid, sorbitanmonolaurate, sorbitan monostearate, and other sorbitan derivatives,polyoxyl derivatives, wax, polyoxyethylene derivatives, sorbitanderivatives; and

viscosity increasing reagents like, agar, alginic acid and its salts,guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose andits derivatives propylene carbonate, polyethylene glycol, hexyleneglycol and tyloxapol.

Formulations described herein also comprise an effective amount ofcrystalline antibody. In particular, the formulations of the inventionmay include a “therapeutically effective amount” or a “prophylacticallyeffective amount” of antibody crystals of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A “therapeutically effective amount” of the antibodycrystals may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the antibody toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Suitable dosages can readily be determined using standard methodology.The antibody is suitably administered to the patient at one time or overa series of treatments. Depending on the above mentioned factors, about1 μg/kg to about 50 mg/kg, as for example 0.1-20 mg/kg of antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily or weekly dosage might range from about 1μg/kg to about 20 mg/kg or more, depending on the condition, thetreatment is repeated until a desired suppression of disease symptomsoccurs. However, other dosage regimens may be useful. In some cases,formulations comprise a concentration of antibody of at least about 1g/L or greater when resolubilized. In other embodiments, the antibodyconcentration is at least about 1 g/L to about 100 g/L whenresolubilized.

Crystals of an antibody, or formulations comprising such crystals, maybe administered alone or as part of a pharmaceutical preparation. Theymay be administered by parenteral, oral or topical routes. For example,they may be administered by oral, pulmonary, nasal, aural, anal, dermal,ocular, intravenous, intramuscular, intraarterial, intraperitoneal,mucosal, sublingual, subcutaneous, transdermal, topical or intracranialroutes, or into the buccal cavity. Specific examples of administrationtechniques comprise pulmonary inhalation, intralesional application,needle injection, dry powder inhalation, skin electroporation, aerosoldelivery, and needle-free injection technologies, including needle-freesubcutaneous administration.

The present invention will now be explained in more detail by means ofthe following, non-limiting, illustrative examples. Guided by thegeneral part of the description and on the basis of his generalknowledge a skilled reader will be enabled to provide furtherembodiments to the invention without undue experimentation.

EXEMPLIFICATION A. Materials a) Protein

Frozen monoclonal antibody (mAb) ABT-874 was obtained from AbbottLaboratories. All experiments were performed from a product lot, wherethe original mAb concentration was 64 mg/mL.

b) Fine Chemicals

Sodium acetate was obtained from Grassing GmbH, Filsum. Polyethyleneglycols of different polymerization grades were obtained from ClariantGmbH, Sulzbach. Furthermore, commercial crystallization screens andreagents (Hampton Research, Nextal Biotechnologies) were used forcertain microscale experiments. All other chemicals were fromSigma-Aldrich, Steinheim, or Merck, Darmstadt.

B. General Methods a) Thawing of ABT-874 Drug Substance

ABT-874 was thawed at 25° C. in agitated water baths.

b) Buffer Exchange—Method A

An aliquot of ABT-874 solution was pipetted into a 30 KDa MWCO Vivaspin20 concentrator (Vivascience). The protein sample was diluted with thenew buffer in a ratio of 1:10, and by centrifugation at 5,000×g at 4° C.(Sigma 4 K 15 lab centrifuge) the sample volume was brought back to theoriginal sample volume. The dilution/centrifugation steps were repeatedonce, resulting in a dilution of 1:100 of the original sample buffer.After adjustment of protein concentration, the solution was sterilefiltered through a 0.2 μm syringe driven filter unit.

b) Buffer Exchange—Method B

An aliquot of ABT-874 solution was placed into a SLIDE-A-LYZER dialysiscassette (Pierce Biotechnology Inc.). The dialysis cassette was placedinto a beaker containing the buffer of choice, and the buffer exchangewas performed at 4° C. overnight with stirring. After adjustment ofprotein concentration, the solution was sterile filtered through a 0.2μm syringe driven filter unit.

c) OD280—Protein Concentration Measurements

A ThermoSpectronics UV1 device was used to assess protein concentrationat a wavelength of 280 nm, applying an extinction coefficient of 1.42cm² mg⁻¹. For this purpose, aliquots of crystallization slurries werecentrifuged at 14,000 rpm, and residual protein concentration wasdetermined in the supernatant.

d) pH Measurements

pH measurements were conducted by using a Mettler Toledo MP220 pH meter.Inlab 413 electrodes and Inlab 423 microelectrodes were utilized.

e) Crystallization Methods

e1) Microscale Crystallization—Sitting Drop Vapor Diffusion Hydra II

Initial crystallization screens were performed using a Hydra IIcrystallization robot and Greiner 96 well plates (three drop wells,Hampton Research). After setting up the plates, the wells were sealedwith Clearseal film (Hampton Research).

e2) Microscale Crystallization—Hanging Drop Vapor Diffusion

Hanging drop vapor diffusion experiments were conducted using VDX plates(with sealant, Hampton Research) and OptiClear plastic cover slides(squares, Hampton Research) or siliconized glass cover slides (circle,Hampton Research), respectively. After preparation of reservoirsolutions, one drop of reservoir solution was admixed with one drop ofthe protein solution on a cover slide, and the well was sealed with theinverted cover slide in such a way that the drop was hanging above thereservoir.

e3) Batch Crystallization—Method A (24 Well Plate)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer (500 μl) in a well. Thewell was subsequently sealed with adhesive tape to prevent waterevaporation.

e4) Batch Crystallization—Method B (Eppendorff Reaction Tube)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer in a 1.5 mL or a 2 mLEppendorff reaction tube.

e5) Batch Crystallization—Method C (Falcon Tubes, No Agitation)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer in a 15 mL or 50 mLFalcon tube.

e6) Batch Crystallization—Method D (Falcon Tubes, Agitation)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer in a 15 mL or 50 mLFalcon tube. Right after closing, the tube was put on a laboratoryshaker (GFL 3013 or GFL 3015) or was alternatively agitated by tumbling.By application of these methods, introduction of stirrers into thesample was avoided.

f) SDS-PAGE

Samples were prepared by adjusting protein concentration to 8 μg/20 μL.The samples were diluted with an SDS/Tris/glycerine buffer containingbromophenol blue. Qualitative SDS PAGE analysis was performed usingInvitrogen NuPage 10% Bis-Tris Gels, NuPage MES SDS Running Buffer andMark12 Wide Range Protein Standards. 20 μL of sample was pipetted into agel pocket. After running the gel and fixation with acetic acid/methanolreagent, staining was performed using the Novex Colloidal Blue StainKit. Gels were dried using Invitrogen Gel-Dry drying solution.

g) Light Microscopy

Crystals were observed using a Zeiss Axiovert 25 or a Nikon Labophotmicroscope. The latter was equipped with a polarization filter set and aJVC TK C1380 color video camera.

h) SE-HPLC

Aggregation levels of ABT-874 samples were assessed by SE-HPLC. A DionexP680 pump, ASI-100 autosampler and UVD170U detector device were used.Aggregated species were separated from the monomer by an AmershamBioscience Superdex 200 10/300 GL gel filtration column, applying avalidated Abbott standard protocol (A-796874.0—ABT 874, J 695).

C. Vapor Diffusion Crystallization Experiments

Concentration values given in the following examples are initial valuesreferring to the antibody solution and the reservoir solution beforemixing of the two solutions.

All pH values, if not described otherwise, refer to the pH of an acetatebuffer stock before it was combined with other substances, like thecrystallization agent.

All buffer molarities, if not described otherwise, refer to sodiumacetate concentrations in a stock solution before pH adjustment,typically performed using acetic acid glacial.

Example 1 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode

A hanging drop vapor diffusion crystallization method was performed onABT-874. ABT-874 was buffered into a buffer containing about 0.1M sodiumacetate at a pH of about 5.2. The protein concentration was adjusted to10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water (fullydesalted and optionally pre-distilled) in each well. In this example,the acetate buffer molarity was kept constant at about 0.1M, and PEG4,000 was varied from about 6% w/v to about 28% w/v in 2% steps. The pHwas about 5.2 throughout. Each condition was assessed in duplicate.About 1 μL of the protein solution was admixed with about 1 μL of aparticular reservoir solution on a square OptiClear plastic cover slide,and the well was sealed with the inverted slide, generating a hangingdrop experiment. The plates were stored at ambient temperature.Microscopy of the drops was performed multiple times during thefollowing thirty days. The conditions were classified into clear drops,drops containing random precipitation, drops containing crystals anddrops containing mixtures of precipitated species and crystals.

RESULTS: From the 24 wells assessed, no crystals were observed.

Example 2 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

A hanging drop vapor diffusion crystallization method was performed onABT-874 at different protein concentration. ABT-874 was buffered into abuffer containing about 0.1M sodium acetate at a pH of about 5.2. Theprotein concentration was adjusted to 50 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 6% w/v to about 28% w/vin 2% steps. The pH was about 5.2 throughout. Each condition wasassessed in duplicate. About 1 μL of the protein solution was admixedwith about 1 μL of a particular reservoir solution on a square OptiClearplastic cover slide, and the well was sealed with the inverted slide,generating a hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

RESULTS: From the 24 wells assessed, crystals were observed at a PEG4,000 concentration of about 16%. The crystals showed needle or needlecluster like morphology.

Example 3 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 400. ABT-874 was buffered into a buffer containingabout 0.1M sodium acetate at a pH of about 5.2. The proteinconcentration was adjusted to 10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG use PEG 400 solution and Milli Q water ineach well. In this example, the acetate buffer molarity was keptconstant at about 0.1M, and PEG 400 was varied from about 30% w/v toabout 40% w/v in 2% steps. The pH was about 5.2 throughout. Eachcondition was assessed in duplicate. About 1 μL of the protein solutionwas admixed with about 1 μL of a particular reservoir solution on asquare OptiClear plastic cover slide, and the well was sealed with theinverted slide, generating a hanging drop experiment. The plates werestored at ambient temperature. Microscopy of the drops was performedmultiple times during the following thirty days. The conditions wereclassified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

RESULTS: From the 12 wells assessed, no crystals were observed.

Example 4 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

A hanging drop vapor diffusion crystallization method was performed onABT-874 at different protein concentration. ABT-874 was buffered into abuffer containing about 0.1M sodium acetate at a pH of about 5.2. Theprotein concentration was adjusted to 50 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG use PEG 400 solution and Milli Q water ineach well. In this example, the acetate buffer molarity was keptconstant at about 0.1M, and PEG 400 was varied from about 30% w/v toabout 40% w/v in 2% steps. The pH was about 5.2 throughout. Eachcondition was assessed in duplicate. About 1 μL of the protein solutionwas admixed with about 1 μL of a particular reservoir solution on asquare OptiClear plastic cover slide, and the well was sealed with theinverted slide, generating a hanging drop experiment. The plates werestored at ambient temperature. Microscopy of the drops was performedmultiple times during the following thirty days. The conditions wereclassified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

RESULTS: From the 12 wells assessed, no crystals were observed.

Example 5 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration and Set Up

A hanging drop vapor diffusion crystallization method was performed onABT-874 using different protein concentration and a different set up.ABT-874 was buffered into a buffer containing about 0.1M sodium acetateat a pH of about 5.2. The protein concentration was adjusted to 50mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 400 solution and Milli Q water in each well.In this example, the acetate buffer molarity was kept constant at about0.1M, and PEG 400 was varied from about 30% w/v to about 40% w/v in 2%steps. The pH was about 5.7 or 6.7, respectively. Each condition wasassessed in duplicate. About 1 μL of the protein solution was admixedwith about 1 μL of a particular reservoir solution on a square OptiClearplastic cover slide, and the well was sealed with the inverted slide,generating a hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following twenty-one days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

RESULTS: From the 24 wells assessed, no crystals were observed.

Example 6 PEG 10,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 10,000. ABT-874 was buffered into a buffer containingabout 0.1M sodium acetate at a pH of about 5.2. The proteinconcentration was adjusted to 10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 10,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 10,000 was varied from about 4% w/v to about 14% w/vin 2% steps. The pH was about 5.2 throughout. Each condition wasassessed in duplicate. About 1 μL of the protein solution was admixedwith about 1 μL of a particular reservoir solution on a square OptiClearplastic cover slide, and the well was sealed with the inverted slide,generating a hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

RESULTS: From the 12 wells assessed, no crystals were observed.

Example 7 PEG 10,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 10,000 and at different protein concentration. ABT-874was buffered into a buffer containing about 0.1M sodium acetate at a pHof about 5.2. The protein concentration was adjusted to 50 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 10,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 10,000 was varied from about 4% w/v to about 14% w/vin 2% steps. The pH was about 5.2 throughout. Each condition wasassessed in duplicate. About 1 μL of the protein solution was admixedwith about 1 μL of a particular reservoir solution on a square OptiClearplastic cover slide, and the well was sealed with the inverted slide,generating a hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

RESULTS: From the 12 wells assessed, no crystals were observed.

Example 8 PEG 4,000/Sodium Acetate Grid Screen In Hanging Drop VaporDiffusion Mode, Different Set Up

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 4,000 and a different set up. ABT-874 was bufferedinto a buffer containing about 0.1M sodium acetate at a pH of about 5.2.The protein concentration was adjusted to 10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 22% w/v to about 28% w/vin 2% steps. The pH was about 4.2, 4.7, 5.2, 5.7, 6.2 and 6.7,respectively. Each condition was assessed in duplicate. About 1 μL ofthe protein solution was admixed with about 1 μL of a particularreservoir solution on a square OptiClear plastic cover slide, and thewell was sealed with the inverted slide, generating a hanging dropexperiment. The plates were stored at ambient temperature. Microscopy ofthe drops was performed multiple times during the following thirty days.The conditions were classified into clear drops, drops containing randomprecipitation, drops containing crystals and drops containing mixturesof precipitated species and crystals.

RESULTS: From the 48 wells assessed, no crystals were observed.

Example 9 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 4,000 and another set up. ABT-874 was buffered into abuffer containing about 0.1M sodium acetate at a pH of about 5.2. Theprotein concentration was adjusted to 10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 8% w/v to about 14% w/vin 2% steps. The pH was about 5.7, 6.2 and 6.7, respectively. Eachcondition was assessed in duplicate. About 1 μL of the protein solutionwas admixed with about 1 μL of a particular reservoir solution on asquare OptiClear plastic cover slide, and the well was sealed with theinverted slide, generating a hanging drop experiment. The plates werestored at ambient temperature. Microscopy of the drops was performedmultiple times during the following thirty days. The conditions wereclassified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

RESULTS: From the 24 wells assessed, crystals were observed at a PEG4,000 concentration of about 10 to 14% at all pH included in thisexample. The crystals showed needle or needle cluster like morphology.

Example 10 PEG 400 Combined with 4,000/Sodium Acetate Grid Screen inHanging Drop Vapor Diffusion Mode

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 400 with 4,000/Sodium Acetate. ABT-874 was bufferedinto a buffer containing about 0.1M sodium acetate at a pH of about 5.2.The protein concentration was adjusted to 10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 8% w/v to about 12% w/vin 2% steps. Simultaneously, PEG 400 was brought into the PEG4,000/acetate solutions at concentrations of about 26% w/v, 28% w/v, 30%w/v and 32% w/v, respectively. The pH was about 5.2 throughout. Eachcondition was assessed in duplicate. About 1 μL of the protein solutionwas admixed with about 1 μL of a particular reservoir solution on asquare OptiClear plastic cover slide, and the well was sealed with theinverted slide, generating a hanging drop experiment. The plates werestored at ambient temperature. Microscopy of the drops was performedmultiple times during the following thirty days. The conditions wereclassified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

RESULTS: From the 24 wells assessed, no crystals were observed.

Example 11 PEG 400 Combined with 4,000/Sodium Acetate Grid Screen inHanging Drop Vapor Diffusion Mode, Different Protein Concentration

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 400 with 4,000/Sodium Acetate with different proteinconcentrations. ABT-874 was buffered into a buffer containing about 0.1Msodium acetate at a pH of about 5.2. The protein concentration wasadjusted to 50 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 4% w/v to about 8% w/vin 2% steps. Simultaneously, PEG 400 was brought into the PEG4,000/acetate solutions and concentrations of about 30% w/v, 32% w/v,34% w/v and 36% w/v, respectively. The pH was about 5.2 throughout. Eachcondition was assessed in duplicate. About 1 μL of the protein solutionwas admixed with about 1 μL of a particular reservoir solution on asquare OptiClear plastic cover slide, and the well was sealed with theinverted slide, generating a hanging drop experiment. The plates werestored at ambient temperature. Microscopy of the drops was performedmultiple times during the following thirty days. The conditions wereclassified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

RESULTS: From the 24 wells assessed, no crystals were observed.

Example 12 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Buffer

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 4,000 with different protein buffers. ABT-874 wasbuffered into a buffer containing about 0.1M sodium acetate at a pH ofabout 5.5. The protein concentration was adjusted to 10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 4% w/v to about 26% w/vin 2% steps. The pH was 5.5 throughout. Each condition was assessed induplicate. About 1 μL of the protein solution was admixed with about 1μL of a particular reservoir solution on a square OptiClear plasticcover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following five days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

RESULTS: From the 24 wells assessed, crystals were observed at a PEG4,000 concentration of about 12% w/v, 18% w/v, 20% w/v, 22% w/v and 24%w/v, respectively. The crystals showed needle or needle cluster likemorphology.

Example 13 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 4,000 with different protein concentrations. ABT-874was buffered into a buffer containing about 0.1M sodium acetate at a pHof about 5.5. The protein concentration was adjusted to 5 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 4% w/v to about 26% w/vin 2% steps. The pH was 5.5 throughout. Each condition was assessed induplicate. About 1 μL of the protein solution was admixed with about 1μL of a particular reservoir solution on a square OptiClear plasticcover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following five days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

RESULTS: From the 24 wells assessed, crystals were observed at a PEG4,000 concentration of about 10% w/v and 14% w/v, respectively. Thecrystals showed needle or needle cluster like morphology.

Example 14 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Buffer

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 4,000/Sodium Acetate with different protein buffer.ABT-874 was buffered into a buffer containing about 0.1M sodium acetateat a pH of about 5.5. The protein concentration was adjusted to 20mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 was varied from about 4% w/v to about 26% w/vin 2% steps. The pH was 5.5 throughout. Each condition was assessed induplicate. About 1 μL of the protein solution was admixed with about 1μL of a particular reservoir solution on a square OptiClear plasticcover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following five days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

RESULTS: From the 24 wells assessed, crystals were observed at a PEG4,000 concentration of about 10% w/v, 14% w/v, 16% w/v, 20% w/v and 22%w/v, respectively. The crystals showed needle or needle cluster likemorphology.

Example 15 Broad Screening of Conditions in Vapor Diffusion Mode

A broad screening hanging drop vapor diffusion crystallization methodwas performed on ABT-874. ABT-874 was buffered into a 20 mM HEPES/150 mMsodium chloride buffer at pH 7.4. The protein concentration was adjustedto 10 mg/mL. In another case, protein concentration was adjusted to 5mg/mL. In another case, protein concentration was adjusted to 20 mg/mL.

Using the Hydra II crystallization roboter, 96 well Greiner plates wereset up at ambient temperature, using several commercially availablecrystallization screens. The protein solution and the crystallizationagent were admixed in a ratio of about 1:1, preferably 1:1.

The following screens were used. Hampton Crystal Screen 1 & 2, HamptonIndex Screen, Hampton SaltRX Screen (all from Hampton Research), NextalThe Classics, The Classics Lite, The PEGs, The Anions, The pH clear andThe Ammonium sulphate (all from Nextal Biotechnologies).

After addition of the protein to the crystallization agent (three dropsper condition, containing the three different protein concentrations asdescribed above), the plates were sealed with Clearseal film. Any platewas set up in quadruplicate and stored at ambient temperature, 4° C.,27° C. and 37° C., respectively. Microscopy of the drops was performedafter six days. The conditions were classified into clear drops, dropscontaining random precipitation, drops containing crystals and dropscontaining mixtures of precipitated species and crystals.

RESULTS: From the 10,368 conditions tested, 4 rendered crystals. Theconditions comprised following protein concentrations andcrystallization agents as declared by the manufacturers:

ambient temperature, ABT-874 at about 20 mg/mL

0.2M ammonium sulphate, 30% w/v PEG 8,000

(Hampton Crystal Screen, C6)

4° C., ABT-874 at about 5 mg/mL

0.1M HEPES pH 7.5, 5% w/v PEG 8,000

(Nextal The Classics Lite, F4)

4° C., ABT-874 at about 10 mg/mL

0.1M HEPES pH 7.5, 5% w/v PEG 6,000, 2.5% v/v MPD

(Nextal The Classics Lite, H9)

4° C., ABT-874 at about 20 mg/mL

0.1M HEPES, 5% w/v PEG 6,000, pH 7.00

(Nextal pH clear, C4)

The crystals showed needle like or needle cluster like morphologies.

Example 16 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 400 with 4,000/Sodium Acetate with a different set up.ABT-874 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer atpH 7.4. The protein concentration was adjusted to 10 mg/mL. In anothercase, protein concentration was adjusted to 5 mg/mL.

A greased VDX plate and circle siliconized glass cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and PEG 4,000 concentration was used at about 12% w/v, 18%w/v, 24% w/v and 30% w/v, respectively. The pH was varied from about 3.6to about 5.6 in 0.2 steps, generating 48 different conditions. Anycondition was set up with the two protein concentrations as describedabove. About 1 μL of the protein solution was admixed with about 1 μL ofa particular reservoir solution on a circle siliconized glass coverslide, and the well was sealed with the inverted slide, generating ahanging drop experiment. The plates were stored at ambient temperature.Microscopy of the drops was performed after six days. The conditionswere classified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

RESULTS: From the 96 conditions tested, crystals in the shape of needleclusters were observed with the 5 mg/mL ABT-874 and about 24% PEG 4,000at pH about 5.6.

Example 17 PEG 4,000/Sodium Citrate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

A hanging drop vapor diffusion crystallization method was performed onABT-874 using PEG 4,000/Sodium Citrate with a different set up. ABT-874was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH 7.4.The protein concentration was adjusted to 10 mg/mL. In another case,protein concentration was adjusted to 5 mg/mL.

A greased VDX plate and circle siliconized glass cover slides were used.500 μL of a particular reservoir solution was prepared by admixingcitrate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the citrate buffer molarity was kept constant atabout 0.1M, and the PEG 4,000 concentration was used at about 12% w/v,18% w/v, 24% w/v or 30% w/v. The pH was varied from about 4.2 to around6.4 in 0.2 steps, generating 48 different conditions. Any condition wasset up with the two protein concentrations as described above. Around 1μL of the protein solution was admixed with around 1 μL of a particularreservoir solution on a circle siliconized glass cover slide, and thewell was sealed with the inverted slide, generating a hanging dropexperiment. The plates were stored at ambient temperature. Microscopy ofthe drops was performed after six days. The conditions were classifiedinto clear drops, drops containing random precipitation, dropscontaining crystals and drops containing mixtures of precipitatedspecies and crystals.

RESULTS: From the 96 conditions tested, no crystals were observed.

D. Batch Crystallization Experiments

A batch crystallization method was performed on ABT-874. Concentrationvalues given in the following examples are initial values referring tothe antibody solution and the crystallization solution before mixing ofthe two solutions.

All pH values, if not described otherwise, refer to the pH of an acetatebuffer stock before it was combined with other substances, like thecrystallization agent.

All buffer molarities, if not described otherwise, refer to sodiumacetate concentrations in a stock solution before pH adjustment,typically performed using acetic acid glacial.

Example 18 PEG 4,000/Sodium Acetate Condition at 1 Ml Batch Volume

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate at 1 Ml batch volume. ABT-874 was buffered into a buffercontaining about 0.1M sodium acetate at a pH of around 5.2. The proteinconcentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 500 μL of theprotein solution with an equal volume of the crystallization buffer in a1.5 mL Eppendorff reaction tube. 500 μL of a particular reservoirsolution was prepared by admixing acetate buffer, 50% w/v PEG 4,000solution and Milli Q water. In this example, the acetate buffer molaritywas 0.1M, and the acetate buffer pH was around 6.7. PEG 4,000 was usedat a concentration of around 14% w/v. The reaction tube was stored atambient temperature. Microscopy of a 1 μL aliquot was performed after 16days.

RESULTS: No crystals were observed after 16 days.

Example 19 PEG 4,000/Sodium Acetate Grid Screen in 300 μL Volume BatchMode

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 300 μL volume batch mode. ABT-874 was buffered into abuffer containing about 0.1M sodium acetate at a pH of around 5.5. Theprotein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 150 μL of theprotein solution with an equal volume of the crystallization buffer in awell. The well plate was subsequently sealed with adhesive tape toprevent water evaporation. 150 μL of a particular reservoir solution wasprepared by admixing acetate buffer, 50% w/v PEG 4,000 solution andMilli Q water in each well. In this example, the acetate buffer molaritywas kept constant at around 0.1M, and the acetate buffer pH was around5.5 throughout. PEG 4,000 was varied from around 12% w/v to around 34%w/v in 2% steps. Any condition was assessed in triplicate. The plate wasstored at ambient temperature. Microscopy of the drops was performedduring the following two days.

RESULTS: From the 36 wells examined, crystals were observed inexperiments, that were set up with between 22% w/v and 26% w/v PEG4,000.

Example 20 PEG 4,000/Sodium Acetate Condition at 1 Ml Batch Volume,Different PEG 4,000 Concentrations

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 1 Ml batch volume using different PEG 4,000 concentrations.ABT-874 was buffered into a buffer containing around 0.1M sodium acetateat a pH of around 5.5. The protein concentration was adjusted to 10mg/mL.

Batch crystallization was performed by admixing around 500 μL of theprotein solution with an equal volume of the crystallization buffer in a1.5 mL Eppendorff reaction tube. 500 μL of a particular reservoirsolution was prepared by admixing acetate buffer, 50% w/v PEG 4,000solution and Milli Q water. In this example, the acetate buffer molaritywas 0.1M, and the acetate buffer pH was around 5.5. PEG 4,000 was usedat a concentration of about 22% w/v. The experiment was set up inquadruplicate. The reaction tubes were stored at ambient temperature.Microscopy of 1 μL aliquots were performed multiple times during thefollowing 78 days. Furthermore, the crystal yield was determined by OD280. An aliquot of the suspension was centrifuged at 14,000 rpm, and theprotein concentration in the supernatant was assessed.

RESULTS: Sword-like crystals appeared after seven days. No precipitatedspecies were observed during the following months of storage. Thecrystal yield as determined by OD280 from residual protein concentrationin the supernatant was between 50 and 70% after sixty days.

Example 21 PEG 4,000/Sodium Acetate Condition at 1 Ml Batch Volume,Different PEG 4,000 Concentrations

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 1 Ml batch volume using different PEG 4,000 concentrations.ABT-874 was buffered into a buffer containing about 0.1M sodium acetateat a pH of about 5.5. The protein concentration was adjusted to 10mg/mL.

Batch crystallization was performed by admixing about 500 μL of theprotein solution with an equal volume of the crystallization buffer in a1.5 mL Eppendorff reaction tube. 500 μL of a particular reservoirsolution was prepared by admixing acetate buffer, 50% w/v PEG 4,000solution and Milli Q water. In this example, the acetate buffer molaritywas 0.1M, and the acetate buffer pH was about 5.5. PEG 4,000 was used ata concentration of about 26% w/v. The reaction tube was stored atambient temperature. Microscopy of a 1 μL aliquot was performed multipletimes during the following months.

RESULTS: After one day, precipitated species were observed. Sword-likecrystals were observed after five days besides the precipitate.

Example 22 PEG 4,000/Sodium Acetate Condition at 1 Ml Batch Volume,Different PEG 4,000 Concentrations

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 1 Ml batch volume using different PEG 4,000 concentrations.ABT-874 was buffered into a buffer containing about 0.1M sodium acetateat a pH of about 5.5. The protein concentration was adjusted to 10mg/mL.

Batch crystallization was performed by admixing about 500 μL of theprotein solution with an equal volume of the crystallization buffer in a1.5 mL Eppendorff reaction tube. 500 μL of a particular reservoirsolution was prepared by admixing acetate buffer, 50% w/v PEG 4,000solution and Milli Q water. In this example, the acetate buffer molaritywas 0.1M, and the acetate buffer pH was about 5.5. PEG 4,000 was used ata concentration of about 24% w/v. The reaction tube was stored atambient temperature. Microscopy of a 1 μL aliquot was performed multipletimes during the following months. Furthermore, the crystal yield wasdetermined by OD 280. An aliquot of the suspension was centrifuged at14,000 rpm, and the protein concentration in the supernatant wasassessed.

RESULTS: Needle cluster like crystals appeared after one day. After fivedays, needle like crystals and platelets were observed besides theneedle cluster like crystals. The crystal yield as determined by OD280from residual protein concentration in the supernatant was between 60and 70% after thirteen days.

Example 23 PEG 4,000/Sodium Acetate Grid Screen in 1 Ml Volume BatchMode, Different Protein Concentration

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 1 Ml batch volume using different protein concentrations.ABT-874 was buffered into a buffer containing about 0.1M sodium acetateat a pH of about 5.5. The protein concentration was adjusted to 5 mg/mL.

Batch crystallization was performed by admixing about 500 μL of theprotein solution with an equal volume of the crystallization buffer in awell. The well plate was subsequently sealed with adhesive tape toprevent water evaporation.

500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant atabout 0.1M, and the acetate buffer pH was about 5.5 throughout. PEG4,000 was varied from about 12% w/v to about 34% w/v in 2% steps. Anycondition was assessed in duplicate. The plate was stored at ambienttemperature. Microscopy of the drops was performed during the followingmonth.

RESULTS: From the 24 wells examined, sword-like crystals were observedin experiments that were set up with about 24% w/v and 26% w/v PEG4,000.

Example 24 PEG 4,000/Sodium Acetate Grid Screen in 1 Ml Volume BatchMode, Different Set Up

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 1 Ml batch volume using different set up. ABT-874 wasbuffered into a buffer containing about 0.1M sodium acetate at a pH ofabout 5.5. The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 500 μL of theprotein solution with an equal volume of the crystallization buffer in awell. The well plate was subsequently sealed with adhesive tape toprevent water evaporation. 500 μL of a particular reservoir solution wasprepared by admixing acetate buffer, 50% w/v PEG 4,000 solution andMilli Q water in each well. In this example, the acetate buffer molaritywas kept constant at about 0.1M, and the acetate buffer pH was about4.1, 4.6 and 5.1, respectively. PEG 4,000 was varied from about 20% w/vto about 28% w/v in 2% steps. The plate was stored at ambienttemperature. Microscopy of the drops was performed during the followingfour days.

RESULTS: From the 18 wells examined, sword-like crystals were observedin experiments that were set up with 28% w/v PEG 4,000 and sodiumacetate buffer at pH 5.1.

Example 25 PEG 4,000/Sodium Acetate Condition at 2 Ml Batch Volume,Different Temperature

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 2 Ml batch volume using different temperatures. ABT-874 wasbuffered into a buffer containing about 0.1M sodium acetate at a pH ofabout 5.5. The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 1 mL of theprotein solution with an equal volume of the crystallization buffer in a2 mL Eppendorff reaction tube. 1 mL of a particular reservoir solutionwas prepared by admixing acetate buffer, 50% w/v PEG 4,000 solution andMilli Q water. In this example, the acetate buffer molarity was 0.1M,and the acetate buffer pH was about 5.5. PEG 4,000 was used at aconcentration of about 22% w/v. The reaction tube was stored at 4-8° C.Microscopy of a 1 μL aliquot was performed multiple times during thefollowing month.

RESULTS: Precipitated species were observed after storage overnight.

Example 26 PEG 4,000/Sodium Acetate Crystallization Condition at 10 MlBatch Volume, Agitation

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 10 Ml batch volume using agitation. ABT-874 was bufferedinto a buffer containing about 0.1M sodium acetate at a pH of about 5.5.The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 5 mL of theprotein solution with an equal volume of the crystallization buffer in a50 mL Falcon tube. 5 mL of the crystallization buffer was prepared byadmixing acetate buffer, 50% w/v PEG 4,000 solution and Milli Q water inthe tube. In this example, the acetate buffer molarity was about 0.1M,and the acetate buffer pH was about 5.5. PEG 4,000 was used at aconcentration of about 24% w/v. The tube was stored at ambienttemperature, agitating the batch on a laboratory shaker. Microscopy of a1 μL aliquot of the solution was performed multiple times during thefollowing weeks.

RESULTS: Sword-like crystals appeared after six days, but were almostcompletely adsorbed to the container surface. It could not be concludedfrom microscopy that the batch was free of precipitated species. Thecrystallization liquor was almost clear.

Example 27 PEG 4,000/Sodium Acetate Crystallization Condition at 10 MlBatch Volume, No Agitation

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 10 Ml batch volume with no agitation. ABT-874 was bufferedinto a buffer containing about 0.1M sodium acetate at a pH of about 5.5.The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 5 mL of theprotein solution with an equal volume of the crystallization buffer in a50 mL Falcon tube. 5 mL of the crystallization buffer was prepared byadmixing acetate buffer, 50% w/v PEG 4,000 solution and Milli Q water inthe tube. In this example, the acetate buffer molarity was about 0.1M,and the acetate buffer pH was about 5.5. PEG 4,000 was used at aconcentration of about 24% w/v. The tube was stored at ambienttemperature. Microscopy of a 1 μL aliquot of the solution was performedmultiple times during the following weeks. Furthermore, the crystalyield was determined by OD 280. An aliquot of the suspension wascentrifuged at 14,000 rpm, and the protein concentration in thesupernatant was assessed.

RESULTS: Needle cluster like crystals appeared after one day. After fourdays, needle like crystals were observed besides the needle cluster likecrystals. The crystal yield as determined by OD280 from residual proteinconcentration in the supernatant was between 30 and 40% after sevendays.

Example 28 PEG 4,000/Sodium Acetate Crystallization Condition at 10 MlBatch Volume, Agitation, Different Container Material

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 10 Ml batch volume using agitation and different containermaterials. ABT-874 was buffered into a buffer containing about 0.1Msodium acetate at a pH of about 5.5. The protein concentration wasadjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 5 mL of theprotein solution with an equal volume of the crystallization buffer in a50 mL glass class I vial. 5 mL of the crystallization buffer wasprepared by admixing acetate buffer, 50% w/v PEG 4,000 solution andMilli Q water in the vial. In this example, the acetate buffer molaritywas about 0.1M, and the acetate buffer pH was about 5.5. PEG 4,000 wasused at a concentration of about 24% w/v. The vial was stored at ambienttemperature, agitating the batch on a laboratory shaker. Microscopy of a1 μL aliquot of the solution was performed multiple times during thefollowing weeks. Furthermore, the crystal yield was determined by OD280. An aliquot of the suspension was centrifuged at 14,000 rpm, and theprotein concentration in the supernatant was assessed.

RESULTS: Sword-like crystals were observed after eighteen days. Thecrystal yield as determined by OD280 from residual protein concentrationin the supernatant was between 40 and 50% after eighteen days. A lightmicroscopic picture of the needle-clusters (width of the picturecorresponding to a length of 450 μm) is shown in FIG. 7.

Example 29 PEG 4,000/Sodium Acetate Crystallization Condition at 10 MlBatch Volume, Agitation, Different Container Material and Influence ofPolysorbate 80

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 10 Ml batch volume using agitation, different containermaterials and influence of polysorbate 80. ABT-874 was buffered into abuffer containing about 0.1M sodium acetate at a pH of about 5.5. Theprotein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 5 mL of theprotein solution with an equal volume of the crystallization buffer in a50 mL glass class I vial. 5 mL of the crystallization buffer wasprepared by admixing acetate buffer, 50% w/v PEG 4,000 solution andMilli Q water in the vial. In this example, the acetate buffer molaritywas about 0.1M, and the acetate buffer pH was about 5.5. PEG 4,000 wasused at a concentration of about 24% w/v. Furthermore, polysorbate 80 ina concentration of 0.1% was added to the buffer. The vial was stored atambient temperature, agitating the batch on a laboratory shaker.Microscopy of a 1 μL aliquot of the solution was performed multipletimes during the following weeks. Furthermore, the crystal yield wasdetermined by OD 280. An aliquot of the suspension was centrifuged at14,000 rpm, and the protein concentration in the supernatant wasassessed.

RESULTS: Sword-like crystals were observed after eighteen days. Nodifference was observed between the crystal shape of this example andExample 28 (no addition of polysorbate 80). The crystal yield asdetermined by OD280 from residual protein concentration in thesupernatant was between 25 and 35% after eighteen days.

Example 30 Different PEG 4,000/Sodium Acetate Crystallization Conditionsat 10 Ml Batch Volume And Comparison Of Agitated And Non AgitatedBatches

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 10 Ml batch volume using a comparison of agitated andnon-agitated batches. ABT-874 was buffered into a buffer containingabout 0.1M sodium acetate at a pH of about 5.5. The proteinconcentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 5 mL of theprotein solution with an equal volume of the crystallization buffer in a50 mL glass class I vial. 5 mL of the crystallization buffer wasprepared by admixing acetate buffer, 50% w/v PEG 4,000 solution andMilli Q water in the vial. In this example, the acetate buffer molaritywas about 0.1M, and the acetate buffer pH was about 5.5. PEG 4,000 wasused at a concentration of about 22% w/v and 24% w/v. The vials werestored at ambient temperature, either without agitation or agitating thebatch by tumbling. Microscopy of a 1 μL aliquot of the solution wasperformed multiple times during the following weeks. Furthermore, thecrystal yield of one batch was determined by OD 280. An aliquot of thesuspension was centrifuged at 14,000 rpm, and the protein concentrationin the supernatant was assessed.

RESULTS: In both agitated batches, precipitated species were observedafter 26 days. The non-agitated batch with the buffer of about 22% w/vPEG 4,000 contained sword-like crystals after 26 days, but the crystalyield was deemed low as the suspension was almost clear macroscopically.The non-agitated batch with the buffer of about 24% w/v PEG 4,000contained sword-like crystals after 26 days. The yield as determinedfrom the supernatant after 70 days was between 65 and 75%.

Example 31 Influence of Seeding

The influence of seeding on ABT-874 crystal yield was examined. Thenon-agitated batch with the crystallization buffer containing about 22%w/v PEG 4,000 from Example 30 showed very low crystal yield after 26days. Therefore, the batch was incubated with about 100 μL of thenon-agitated batch with the crystallization buffer containing about 24%w/v PEG 4,000 from the same example.

RESULTS: No obvious yield extension resulted from the incubation withseed crystals.

Example 32 PEG 4,000/Sodium Acetate Crystallization Conditions at 10 MlBatch Volume, Different Protein Concentration, Comparison of Agitatedand Non Agitated Batches

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 10 Ml batch volume using different protein concentrationsand a comparison of agitated and non-agitated batches. ABT-874 wasbuffered into a buffer containing about 0.1M sodium acetate at a pH ofabout 5.5. The protein concentration was adjusted to 5 mg/mL.

Batch crystallization was performed by admixing about 5 mL of theprotein solution with an equal volume of the crystallization buffer in a15 mL glass class I vial. 5 mL of the crystallization buffer wasprepared by admixing acetate buffer, 50% w/v PEG 4,000 solution andMilli Q water in the vial. In this example, the acetate buffer molaritywas about 0.1M, and the acetate buffer pH was about 5.5. PEG 4,000 wasused at a concentration of about 22% w/v, 24% w/v and 26% w/v. The vialswere stored at ambient temperature, either without agitation or withagitating the batch on a laboratory shaker. Microscopy of a 1 μL aliquotof the solution was performed multiple times during the following weeks.Furthermore, the crystal yield of one batch was determined by OD 280. Analiquot of the suspension was centrifuged at 14,000 rpm, and the proteinconcentration in the supernatant was assessed.

RESULTS: The batches containing the buffer with about 22% w/v and about24% w/v PEG 4,000 were clear after 65 days. While the agitated batchcontaining the crystallization buffer with about 26% w/v PEG 4,000contained precipitated species after 4 days, the non-agitated batch ofthe same crystallization buffer contained sword-like crystals after 4days. The crystal yield of this particular batch as determined from thesupernatant after 26 days was between 40 and 50%. A light microscopicpicture of the crystals (width of the picture corresponding to a lengthof 225 μm) obtained without agitation is shown in FIG. 8.

Example 33 PEG 4,000/Sodium Acetate Crystallization Condition at 10 MlBatch Volume, Different Set Up

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 10 Ml batch volume using a different set up. ABT-874 wasbuffered into a buffer containing about 0.1M sodium acetate at a pH ofabout 5.5. The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 5 mL of theprotein solution with an equal volume of the crystallization buffer in a15 mL Falcon tube. 5 mL of the crystallization buffer was prepared byadmixing acetate buffer, 50% w/v PEG 4,000 solution and Milli Q water inthe tube. In this example, the acetate buffer molarity was about 0.1M,and the acetate buffer pH was about 5.5. PEG 4,000 was used at aconcentration of about 22% w/v. The tube was stored at ambienttemperature. Microscopy of a 1 μL aliquot of the solution was performedmultiple times during the following weeks. Furthermore, the crystalyield of the batch was determined by OD 280. An aliquot of thesuspension was centrifuged at 14,000 rpm, and the protein concentrationin the supernatant was assessed.

RESULTS: Sword-like crystals were observed after 11 days. The crystalyield of this batch as determined from the supernatant after 26 days wasbetween 40 and 50%. A light microscopic picture of the crystals (widthof the picture corresponding to a length of 450 μm) obtained withoutagitation after 26 days is shown in FIG. 9.

Example 34a PEG 4,000/sodium acetate crystallization condition at 50 mLBatch Volume

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 50 mL batch volume. ABT-874 was buffered into a buffercontaining about 0.1M sodium acetate at a pH of about 5.5. The proteinconcentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 25 mL of theprotein solution with an equal volume of the crystallization buffer in a50 mL Falcon tube. 25 mL of the crystallization buffer was prepared byadmixing acetate buffer, 50% w/v PEG 4,000 solution and Milli Q water inthe tube. In this example, the acetate buffer molarity was about 0.1M,and the acetate buffer pH was about 5.5. PEG 4,000 was used at aconcentration of about 22% w/v. The tube was stored at ambienttemperature. Microscopy of a 1 μL aliquot of the solution was performedmultiple times during the following weeks. Furthermore, the crystalyield of the batch was determined by OD 280. An aliquot of thesuspension was centrifuged at 14,000 rpm, and the protein concentrationin the supernatant was assessed.

RESULTS: Sword-like crystals were observed after 3 days. The crystalyield of this batch as determined from the supernatant after 16 days wasbetween 50 and 60%.

Example 34b PEG 4,000/Sodium Acetate Crystallization Condition at 700 MlBatch Volume

A crystallization method was performed on ABT-874 using PEG 4,000/SodiumAcetate in a 700 mL batch volume. ABT-874 was buffered into a buffercontaining about 0.1M sodium acetate at a pH of about 5.5. The proteinconcentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing about 350 mL of theprotein solution with an equal volume of the crystallization buffer in a1 L poly propylene bottle. 350 mL of the crystallization buffer wasprepared by admixing acetate buffer, PEG 4,000 and Milli Q water. Inthis example, the acetate buffer molarity was about 0.1M, and theacetate buffer pH was about 5.5. PEG 4,000 was used at a concentrationof about 22% w/v. The bottle was stored at ambient temperature.Microscopy of a 1 μL aliquot of the solution was performed after 40days. Furthermore, the crystal yield of the batch was determined by OD280. An aliquot of the suspension was centrifuged at 14,000 rpm, and theprotein concentration in the supernatant was assessed.

RESULTS: Sword-like crystals were observed after 40 days. The crystalyield of this batch as determined from the supernatant after 40 days wasbetween 50 and 60%. A light microscopic picture of the crystals (widthof the picture corresponding to a length of 450 μm) obtained after 40days without agitation is shown in FIG. 10.

The experimental conditions of the above batch experiments aresummarized in the following Table 1:

TABLE 1 Batch Experiments Batch Protein day of Volume, CrystallizationBuffer Crystals pH pH Conc. Final visual Example ml (initialconcentrations) Agitation (Yield %) Bufer Final mg/ml Temp. control 18 114%¹ PEG4000, 0.1 M NaAc — 6.7 5 amb 16 d 25 2 22% PEG4000, 0.1 M NaAcprecip. 5.5 5.6 5 4-8° C.  1 d 19 0.3 22-26% PEG4000, 0.1 M NaAc +(n.d.)5.5 5 amb  2 d 20 1 22% PEG4000, 0.1 M NaAc +(50-70) 5.5 5.6 5 amb  7 d21 1 26% PEG4000, 0.1 M NaAc +(n.d.) 5.5 5.6 5 amb  5 d 22 1 24%PEG4000, 0.1 M NaAc +(60-70) (13 d) 5.5 5.6 5 amb  1 d 23 1 24-26%PEG4000, 0.1 M NaAc +(n.d.) 5.5 5.6 2.5 amb  2 d 24 1 28% PEG4000, 0.1 MNaAc +(n.d.) 5.1 5.2-5.3 5 amb  4 d 26 10 24% PEG4000, 0.1 M NaAc ++(n.d.) 5.5 5.6 5 amb  6 d 27 10 24% PEG4000, 0.1 M NaAc +(30-40) 5.55.6 5 amb  1 d 28 10 24% PEG4000, 0.1 M NaAc + +(40-50) 5.5 5.6 5 amb 18d 29 10 24% PEG4000, 0.1 M NaAc + +(25-35) 5.5 5.6 5 amb 18 d 0.1%polysorbate 80 30 10 22% PEG4000, 0.1 M NaAc +(n.d.) 5.5 5.6 5 amb 26 d22% PEG4000, 0.1 M NaAc + precip. 24% PEG4000, 0.1 M NaAc +65-75 (70d)26 d 24% PEG4000, 0.1 M NaAc + precip. 32 10 22% PEG4000, 0.1 M NaAcnone 5.5 5.6 2.5 amb 64 d 24% PEG4000, 0.1 M NaAc none 64 d 26% PEG4000,0.1 M NaAc +(40-50)  4 d 26% PEG4000, 0.1 M NaAc + precip.  4 d 33 1022% PEG4000, 0.1 M NaAc +(40-50) (26 d) 5.5 5.6 5 amb 11 d 34a 50 22%PEG4000, 0.1 M NaAc +(50-60) (16 d) 5.5 5.6 5 amb  3 d 34b 700 22%PEG4000, 0.1 M NaAc +(50-60) (40 d) 5.5 5.6 5 amb 40   ¹% (w/v)

E. Methods for Crystal Processing and Analysis Example 35 Washing ofCrystals

After formation of the crystals, a washing step without redissolving thecrystals may be favorable. After the crystallization process wasfinished, the crystal slurry was transferred into a centrifugation tubeand centrifuged at 500 to 1000×g for twenty minutes. The centrifugationwas performed at 4° C. or ambient temperature. After centrifugation, thesupernatant was decanted, and the crystal pellet were easily resuspendedin a buffer containing about 24% w/v PEG 4,000 in about 0.1M sodiumacetate at a pH about 5.5. No measurable solubility of ABT-874 crystalsin such a washing buffer occurred, as analyzed by OD280. Thecentrifugation/resuspension steps were subsequently repeated for one tothree times, and after this washing procedure, the pellet wasresuspended and stored in such a buffer.

Example 36 Analysis of Crystals by SDS PAGE

To confirm the protein character of the crystals, the crystals werewashed with a washing buffer as described in example 32. After assuringby OD280 that no more dissolved protein was in the liquor, the crystalswere centrifuged, the supernatant was decanted, and the crystals weresubsequently dissolved in distilled water. OD280 measurement of thissolution revealed that protein was now present, as the absorbance of thesample was now significantly higher as in the residual washing buffer.SDS PAGE analysis of this solution of redissolved crystals, whencompared to an original ABT-874 sample, showed the same pattern.

Example 37 Analysis of Crystals by SE-HPLC

To assess the content of aggregated species of the ABT-874 crystals, analiquot of washed crystals was centrifuged and redissolved in theSE-HPLC running buffer (92 mM di sodium hydrogen phosphate/211 mM disodium sulfate pH 7.0). Right after the end of the crystallizationprocess, in this example 16 days at ambient temperature, the aggregatecontent typically increased slightly from about 0.9% to about 1.6-1.7%.It is not yet clear whether such aggregates are contained in thecrystals or at their surface and were not properly removed by thewashing process.

F. Miscellaneous Examples

Concentration values given in the following examples are initial valuesreferring to the antibody solution and the crystallization solutionbefore mixing of the two solutions.

All pH values, if not described otherwise, refer to the pH of an acetatebuffer stock before it was combined with other substances, like thecrystallization agent.

All buffer molarities, if not described otherwise, refer to sodiumacetate concentrations in a stock solution before pH adjustment,typically performed using acetic acid glacial.

Example 38 Solid Crystallization Agent

ABT-874 was buffered into a buffer containing about 0.1M sodium acetateat a pH of about 5.5. The protein concentration was adjusted to 10mg/mL.

Batch crystallization was performed by admixing about 500 μL of theprotein solution with about 380 μL acetate buffer (0.1M, pH 5.5) in a 2mL Eppendorf reaction tube. Subsequently, solid polyethylene glycol wasadded to a final concentration of 12% m/v (120 mg/mL). The tube wassubsequently closed and agitated until complete dissolution of thecrystallization agent. The tube was stored at ambient temperaturewithout agitation. Microscopy of aliquots of the crystallization mixturewas performed multiple times during the following weeks.

RESULTS: Sword-like crystals were observed after seven days.

Example 39 Different Buffer Preparation Protocol and Preparation ofCrystals

In this example, the acetate buffers were prepared as described in thefollowing: 60 g of acetic acid glacial were diluted with about 840 mL ofpurified water. The pH was adjusted with sodium hydroxide solution andthe volume adjusted to 1,000 mL. In this case, the total acetate amountwas fixed at 1M (100 mM in the protein solution, the crystallizationbuffer and the crystallization mixture).

Crystallization is performed as according to Example 34a; sword-likecrystals are observed after three days.

Example 40 Preparation of Encapsulated Crystals

Crystals as obtained in Example 34 are positively charged as determinedvia zeta potential measurement using a Malvern Instruments Zetasizernano. The crystals are washed and suspended in a buffer containingexcipients which conserve crystallinity, and which has a pH that keepsthe crystals charged. Subsequently, an appropriate encapsulating agentis added to the crystal suspension. In this context, an appropriateencapsulating agent is a (polymeric) substance with low toxicity,biodegradability and counter ionic character. Due to this counter ioniccharacter, the substance is attracted to the crystals and allowscoating. By this technique, the dissolution of crystals in media, whichdo not contain any other excipient maintaining crystallinity ispreferably sustained.

Example 41 Preparation of Encapsulated/Embedded Crystals

Crystals are obtained as described in Example 34. The crystals arewashed and suspended in a buffer containing excipients which conservecrystallinity.

The crystals can then be embedded by drying the crystals and combiningthese dried crystals with a carrier, e.g. by compression, meltdispersion, etc.

-   -   encapsulated/embedded by combining a crystal suspension with a        carrier solution which is not miscible with water. The carrier        precipitates after removal of the solvent of the carrier.        Subsequently, the material is dried.    -   encapsulated/embedded by combining a crystal suspension with a        water miscible carrier solution. The carrier precipitates as its        solubility limit is exceeded in the mixture.    -   embedded by combining dried crystals or a crystal suspension        with a water miscible carrier solution.    -   embedded by combining dried crystals with a carrier solution        which is not water miscible.

Example 42 Investigation of Precipitated ABT-874 a) Precipitation

Acetate buffer was prepared by dissolving 1 mole of sodium acetate inwater and adjusting pH to 5.5 with acetic acid (100%). The stocksolution was diluted 1:10 with water for buffer exchange. The PEG 4000solution was prepared by dissolving 20 g PEG 4000 in 5 mL 1M sodiumacetate buffer pH 5.5 and water. After dissolution, the volume wasadjusted to 50 mL with water. 5 mL of 10 mg/mL ABT874 (in 0.1M sodiumacetate buffer pH 5.5) (original buffer exchanged by diafiltration) wereadmixed with 5 mL 40% PEG 4000 in 0.1M sodium acetate buffer pH 5.5.

The precipitate batch was kept at room temperature overnight withoutagitation. Non-birefringent particles in the magnitude of approx. 1-10μm formed.

b) Washing of Precipitate

2 mL of the precipitate slurry were put into a centrifuge andcentrifuged at 500×g for 20 min. The supernatant was discarded, and thepellet was resuspended in 2 mL of a 40% PEG 4000 in 0.1M sodium acetatebuffer pH 5.5 (prepared in accordance to the procedure above). Proteinconcentration of the final suspension was determined by OD280 to be 3.9mg/mL.

G. Crystal Characterization

In the following section, experiments that were performed to determinewhether crystalline monoclonal antibody ABT-874 retains the bioactivitycharacteristic of never-crystallized ABT-874 upon redissolution of thecrystalline material are summarized.

G1. Bioactivity Test by Determination of the IFN-γ Production of NK-92Cells a) General Method

The biological activity of redissolved ABT-874 crystals was measured bya cell-based assay that monitors the IFN-γ production of NK-92 cells inresponse to stimulation by IL-12. Prior to analysis the samples werediluted first to 30 μg/mL in cell culture medium (α-MEM medium with 20%FCS and 200 mM L-glutamine). Subsequently samples were further dilutedin 11 steps from 3 μg/mL to 0.1 ng/mL. The IL-12 solution was diluted to10 ng/mL in cell culture medium and added to the ABT-874 samples. Themixtures were then incubated at 37° C. and 5% CO2 for 1 hour.

A suspension of NK-92 cells (2.0×106 cells/mL) was pipetted into a96-well microplate, the ABT-874/IL-12 mixtures were added to the cellsand the microplates were then incubated at 37° C. and 5% CO2 for about20 hours. After incubation the microplates were centrifuged at 1,000 rpmand 5° C. for 10 min and 50 μl of the supernatant of each well were usedto measure the amount of IFN-γ produced by the cells by an ELISA (ELISAKit Human Interferon-γ, Pierce, Cat. No. EHIFNG).

The biotinylated anti IFN-γ antibody solution was pipetted into the96-well precoated microplate and the cell culture supernatants wereadded (4 rows for each of both samples). After incubation of themicroplate for 2 hours at room temperature it was washed. After this theStreptavidin-HRP solution was added and the microplate was incubated foranother 30 min and then washed. After the TMB substrate was added, themicroplate was incubated at room temperature for about 20 min in thedark and the reaction was then stopped by adding the stop solution.

Finally the absorption was measured within the next 5 min in amicroplate reader at 450 nm (correction wavelength 550 nm) and theresults were plotted versus the ABT-874 concentration. The IC50 valueswere then assessed using a 4-parameter nonlinear curve fit and therelative biological activity of the sample was calculated by dividingthe IC50 value of the reference standard by the IC50 value of the sampleand multiplication by 100%.

b) Relative Activity for ABT-874 Crystals

The test was performed as a comparison of the biological activity of thesample to that of a reference standard. The amounts of IFN-γ produced bythe cells were measured by a commercially available ELISA kit and werereported as absorption units at a wavelength of 450 nm. These values,plotted versus the concentration of ABT-874 and assessed by a4-parameter nonlinear regression, revealed the IC50 values for theinhibition of the IL-12 effect by ABT-874. Since both samples were runin four repeats on one microplate this results in four IC50 values forABT-874 reference standard and the sample respectively. Subsequently,the mean of the IC50 values of the reference standard was calculated andthe relative activity of each repeat of the sample was assessed bydividing the mean IC50 value of the reference standard by the relevantIC50 value of the sample and multiplication by 100%.

The test of the sample (crystal suspension 2.9 mg/mL) revealed arelative biological activity of 98%. Thus, the sample can be consideredas fully biologically active.

G2. Microscopic Characterization

In the following, data on microscopic characterization of crystals ofABT-874 will be presented.

a) Optical Analysis of mAb Crystal Batch Samples

After homogenization, aliquots of 1 to 10 μL sample volume were pipettedonto an object holder plate and were covered with a glass cover slide.The crystal preparations were assessed using a Zeiss Axiovert 25inverted light microscope equipped with E-PI 10× oculars and 10×, 20×and 40× objectives, respectively. Pictures were taken using a digitalcamera (Sony Cybershot DSC S75).

b) Scanning Electron Microscope (SEM) characterization of ABT-874crystals

To image protein crystals with an electron microscope they must be dry,electrically conductive and stable enough to tolerate high vacuum andthe energy of an electron beam. This protocol separates the crystalsfrom their buffer by filtration, stabilizes the crystals by chemicallyfixing them with a glutaraldehyde based fixative, dehydrates themthrough a graded series of ethanol, dries them by the critical pointmethod and plasma coats them with gold to make them electricallyconductive.

b1) Materials

-   -   0.2 M Sorensen's Phosphate Buffer (SPB)—0.15 M disodium        phosphate, 0.05 M monobasic potassium phosphate, pH 7.3    -   Karnovsky's fixative—2.5% glutaraldehyde, 1.5% paraformaldehyde,        0.1 M SPB    -   50%, 75%, 95% and 100% ethanol    -   ABT-874 crystal sample in crystallization buffer (from Example        34, stored in washing buffer from Example 35)    -   ABT-874 crystallization buffer (washing buffer from example 35)    -   Millipore stainless steel filter assembly for attaching 13 mm        filter membranes to syringes    -   0.4 μm polycarbonate filter membranes (Nucleopore, Cat#110407)        b2) Equipment    -   Critical Point Dryer (CPD)—Baltec Model CPD030, Asset LC978501    -   Scanning electron microscope (SEM)—Philips XL30 field emission        scanning electron microscope    -   Sputter Coater—Denton Desk II sputter coater, Asset LC827847        b3) Procedure

Steps 3-12 are performed by flushing solution through the filterassembly and holding the syringe on the filter assembly for designatedhold time.

-   -   1. Load syringe filter holder with polycarbonate filter;    -   2. Mix 0.1 ml of crystal sample with 0.4 ml of crystal buffer in        1.0 ml syringe;    -   3. Dispense diluted crystal solution through filter assembly;    -   4. Dispense 1 ml of crystal buffer and hold for 2 min;    -   5. Dispense 1 ml of 50% fix, 50% crystal buffer and hold for 2        min;    -   6. Dispense 1 ml of 100% fixative and hold for 2 min;    -   7. Dispense 1 ml of SPB and hold for 2 min;    -   8. Dispense 1 ml of SPB and hold for 2 min, again;    -   9. Dispense 1 ml of 50% ethanol and hold for 2 min;    -   10. Dispense 1 ml of 75% ethanol and hold for 2 min;    -   11. Dispense 1 ml of 95% ethanol and hold for 2 min;    -   12. Dispense 1 ml of 100% ethanol and hold for 2 min, repeat        step 3 times;    -   13. Transfer filter membrane with attached crystals to CPD        filled w/100% ethanol;    -   14. Process filter through CPD as follows:        -   a. Five exchanges of liquid CO2 at 10° C., mixing for 5            minutes per exchange;        -   b. Heat to 40° C., 80 bar pressure; and        -   c. Slowly bleed back to atmosphere over 20 minutes;    -   15. Mount filter membrane on SEM support;    -   16. Sputter coat w/gold for 60 seconds;    -   17. Examine with SEM;

c) Results

In the attached FIGS. 1 to 5 representative pictures of ABT-874 crystalsare presented.

FIG. 1 shows a light micrograph of ABT-874 crystals in crystallizationbuffer (from Example 34, stored in washing buffer from example 35)obtained according to Example 34. The crystal habit is similar to habitof fixed dried crystals shown in FIGS. 2 to 5. The crystals exhibitedbirefringence.

FIGS. 2 to 5 show SEMs at different magnification of ABT-874 crystalsobtained according to Example 34.

G3. Birefringence

Crystals as generated from all batch experiments exhibitedbirefringence.

G4. Syringeability.

An ABT-874 crystal suspension of 150 mg/mL protein incorporated incrystals and formulated in a washing buffer from example 35 issyringeable through a 27 G needle

H. Capillary Isoelectric Focusing (cIEF) Experiments with ABT-874 a)Equipment

The iCE280 analyzer (Convergent Bioscience) was used for the analysis.System ID 1054 (IS #2785).

b) Material

The capillary used was of 50 mm length, 100 μm ID column, coated(Convergent, Catalogue #101700. The Electrolytes used were—Anolyte (80mM H₃PO₄) and Catholyte (100 mM NaOH). (Convergent, Catalogue #101800).Carrier ampholyte is 4% Pharmalyte (8-10.5), (GE Healthcare, Catalogue#17-0455-01. Additive was methyl cellulose (0.35%), (Convergent,Catalogue #101876). Internal pl markers were from BioRad (8.4, 8.5, 10.1and 10.4—BioRad, Catalogue number 148-2100, Lot#482-511) pl marker mix.

Volume (μL) PI marker 8.4 2.5 PI marker 8.5 2.5 PI marker 10.1 2.5 PImarker 10.4 2.5 Water 40 Total 50

c) Methods

Focusing time was 2 minute at 1500V and 20 minutes at 3000V. Samplepreparation procedure—Mab crystals, Mab precipitate and the referencestandard were all diluted to about 1 mg/ml in Milli-Q water. Samplepreparation procedure (with urea).

Volume (μL) Milli-Q water 92 1% Methyl cellulose 70 Carrier—Pharmalyte 8Sample (1 mg/mL) 30 PI marker mix (Table 1) 16 Total 216

The samples were mixed in 1.5 mL micro-centrifuge tubes as shown in thetable above. Urea was then added (20 mg) to give a final concentrationof about 1.6 M. The centrifuge tubes were then vortexed, centrifuged for10 minutes and then carefully transferred into vials for analysis.

d) Results

The following samples were analyzed:

ABT-874 crystal buffer (washing buffer from example 35)ABT-874 crystals (obtained according to Example 33, in washing bufferfrom example 35)Reference Standard (ABT-874 liquid sample)The results are shown in the attached FIGS. 6A to C.

Example 43 Retention of Native Secondary Structure UponCrystallization/Redissolution of Crystals

IR spectra were recorded with a Confocheck system on a Bruker OpticsTensor 27 according to manufacturers instructions. Liquid samples wereanalyzed using a MicroBiolytics AquaSpec cell. Measurements of proteinsuspensions were performed with a Harrick BioATRII Cell™. Each samplewas assessed by performing at least two measurements of 120 to 500 scansat 25° C. Blank buffer spectra were subtracted from the protein spectra,respectively. Protein second derivative spectra were generated byFourier transformation and vector normalised from 1580-1720 cm⁻¹ forrelative comparison.

Redissolution of crystals was performed as follows. Crystal suspensionswere centrifuged, the supernatant discarded, and the crystal pellet wasdissolved in 0.1 M sodium acetate buffer pH 5.5 to 10 mg/mL proteinconcentration.

FIG. 11 depicts FT-IR second derivative spectra of crystalline ABT-874suspensions, which were crystallized following the process as describedin Example 34b, washed following the procedure introduced in Example 35,and redissolved. The spectra demonstrate that no significant alterationsof the secondary structure were observed, either in the crystallinesolid state or after redissolution.

Example 44 Stability Data (SE HPLC, FT-IR, Morphology)

ABT-874 was crystallized using the crystallization procedure describedin Example 34b. The crystals were washed as described in Example 35,with a dispersion buffer containing 22% PEG 4,000 and 0.1 M sodiumacetate and the pH was adjusted to 5.5 with acetic acid glacial.Subsequently, the crystals were concentrated to 5 mg/mL and 50 mg/mLprotein by centrifugation, respectively, and stored at 2-8° C.

Stability data of 5 mg/ml and 50 mg/mL crystalline ABT-874 over 3 monthsstorage at 2-8° C. indicated retention of above 90% monomer.

(a) SE-HPLC

TABLE 2 Stability Data of 5 mg/mL crystalline ABT-874 afterredissolution Time point Aggregates (%) Monomer (%) Fragments (%) T0 3.995.8 0.3 1 months 5.7 94.0 0.3 3 months 8.7 91.0 0.3

TABLE 3 Stability Data of 50 mg/mL crystalline ABT-874 afterredissolution Time point Aggregates (%) Monomer (%) Fragments (%) T0 3.896.0 0.2 1 months 4.6 95.0 0.4 3 months 6.3 93.4 0.3

A Dionex HPLC system (P680 pump, ASI 100 autosampler, UVD170U) was usedto measure stability of the ABT-874 antibody. ABT-874 samples wereseparated on a GE Superdex® 200 column, applying a flow rate of 0.75mL/min. Detection was carried out at a wavelength of 214 nm. The runningbuffer consisted of 0.2 M di sodium sulphate in 0.09 M sodium phosphatebuffer, pH 7.0.

(b) FT-IR

IR spectra were recorded with a Confocheck system on a Bruker OpticsTensor 27. Liquid samples were analyzed using a MicroBiolytics AquaSpeccell. Measurements of protein suspensions were performed with a HarrickBioATRII Cell™ Each sample was assessed by performing at least twomeasurements of 120 to 500 scans at 25° C. Blank buffer spectra weresubtracted from the protein spectra, respectively. Protein secondderivative spectra were generated by Fourier transformation and vectornormalised from 1580-1720 cm⁻¹ for relative comparison.

Redissolution of crystals was performed as follows. Crystal suspensionswere centrifuged, the supernatant discarded, and the pellet wasdissolved in 0.1 M sodium acetate buffer pH 5.5 to 10 mg/mL proteinconcentration.

FIG. 2 depicts FT-IR second derivative spectra of crystalline ABT-874suspensions (50 mg/mL shelf stability samples, prepared as describedabove and stored for 3 months at 25° C.) and after redissolution of suchpre-treated crystals. The spectra demonstrate that no significantalterations of the secondary structure were observed upon storage at 25°C. for three months, either in the crystalline solid state or afterredissolution.

(c) Morphology

After 3 months storage at 2-8° C., no significant morphological changewas observed in light microscopy analysis of the crystals. Aliquots of 1to 10 μL sample volume were pipetted onto an object holder plate,diluted with formulation buffer (22% PEG) and covered with a glass coverslide. The preparations were assessed using a Zeiss Axiovert 25 invertedlight microscope equipped with E-PI 10× oculars and 10×, 20× and 40×objectives, respectively.

Example 45 Yield Extension of the Crystallization Process

The endpoint of a crystallization process can be defined as the timepoint when OD₂₈₀ measurements of aliquots of the supernatant of thecrystallization slurry are constant, e.g., for three subsequent days. Ayield extension is possible by adding a certain amount of additional PEG4,000 (50% w/v solution in around 0.1 M sodium acetate buffer at a pH ofaround 5.5) to the supernatant of the crystallization slurry. Crystalsthat are similar to the first crop form during the following days.Applying this procedure, the overall yield is easily driven beyond 90%,without the introduction of precipitation.

For example, the PEG 4,000 concentration is raised from around 11% w/vto around 22% w/v, around 20% w/v, around 18% w/v, around 16% w/v, oraround 14% w/v, in aliquots of the supernatant of Example 34b. Afterstorage for several days at ambient temperature (e.g., between about 20and about 25° C.), precipitated species are observed at certain PEG4,000 concentrations, e.g., around 22% w/v, around 20% w/v or around 18%w/v PEG 4,000. Crystals without concomitant precipitation are found atlower PEG 4,000 concentrations, e.g., at around 16% w/v and around 14%w/v PEG 4,000. By adding PEG 4,000 to an overall concentration of, e.g.,around 14% w/v to the residual supernatant of the crystallizationslurry, the overall crystal yield is driven from around 60% to around70% to over 90% in a few days.

Example 46 Yield Extension Applying a Continuous Process

In this example, additional precipitant and/or protein is “titrated” toa crystallization batch (optionally containing a certain amount ofcrystallization agent) at a predefined rate. Continuous crystallizationover time is induced, finally resulting in over 90% crystal yield.

Example 47 Seeding of ABT-874 Crystallization Batches

Spontaneous nucleation is statistic in nature. Seeds, which mightconsist of the same protein (homogeneous seeding) or another substance(heterogeneous seeding) than the one being crystallized, provide atemplate on which further molecules can assemble. Thus, seeding maythereby accelerate crystallization.

An ABT-874 crystallization batch was prepared as described in Example34b. After mixing the protein solution with the crystallization buffer,the mixture was seeded by homogeneous seeding with ABT-874 crystals. Forexample, an aliquot of a crystal suspension prepared as described inExample 34b, exhibiting around 50 to 60% crystal yield, was added, e.g.,in a 1/20 ratio (v/v) to the crystallization batch. Applying thisstrategy, total crystal yields and process durations were furtheroptimized towards higher yields in shorter process times.

Briefly, an ABT-874 crystallization mixture (5 mg/mL protein and 11% PEG4,000 in 0.1M acetate buffer pH 5.5) was prepared and divided into two40 mL aliquots. The first batch was stored at RT without furtherprocedures and the second batch was seeded by adding 2 mL of acrystallization mixture of the same composition that already exhibited65% of crystal yield (6.5 mg seeds, calculated on the base ofcrystallized protein, in comparison to 200 mg ABT-874 in the batch). Theplots depicted in FIG. 13 illustrate that by applying this seedingapproach, the overall yield was extended by around 15% within 80 days,whereas the parallel curve progression suggested that process times toreach maximum yield were not significantly reduced. FIG. 13 suggeststhat although the non-seeded batch reached a plateau of yield afteraround 80 days, the theoretically possible yield might be as high as forthe seeded batch, meaning that seeding reduced the duration of thecrystallization process rather than extending the yield.

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety, as are the references cited therein. The practice of thepresent invention will employ, unless otherwise indicated, conventionaltechniques of crystallization and formulation, which are well known inthe art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

1. A batch crystallization method for crystallizing an anti-human IL-12antibody, the method comprising the steps of: (a) providing an aqueoussolution of the antibody in admixture with at least one polyalkyleneglycol as crystallization agent; and (b) incubating the aqueouscrystallization mixture until crystals of the antibody are formed. 2.The crystallization method according to claim 1, wherein the pH of theaqueous crystallization mixture is in the range of about pH 4 to about6.5.
 3. The crystallization method according to claim 1, wherein theaqueous crystallization mixture comprises a buffer.
 4. Thecrystallization method according to claim 3, wherein the buffercomprises an acetate buffer.
 5. The crystallization method according toclaim 4, wherein the buffer comprises sodium acetate.
 6. Thecrystallization method according to any one of claims 3 to 5, whereinthe buffer concentration in the aqueous crystallization mixture is up toabout 0.5 M.
 7. The crystallization method according to any one ofclaims 1-5, wherein the polyalkylene glycol has an average molecularweight in the range of about 400 to about 10,000.
 8. The crystallizationmethod according to claim 7, wherein the polyalkylene glycol ispolyethylene glycol.
 9. The crystallization method according to any oneof claims 1-5, wherein the polyalkylene glycol concentration in thecrystallization mixture is in the range of about 5 to 30% (w/v).
 10. Thecrystallization method according to claim 9, wherein the polyalkyleneglycol is polyethylene glycol.
 11. The crystallization method accordingto any one of claims 1-5, wherein at least one of the followingadditional crystallization conditions are met: a) incubation isperformed for between about 1 hour to about 250 days; b) incubation isperformed at a temperature between about 4° C. and about 37° C.; c) theantibody concentration is in the range of about 0.5 to about 280 mg/ml.12. The crystallization method according to claim 1, further comprisingthe step of drying the crystals.
 13. The crystallization methodaccording to any one of claims 1-5, further comprising the step ofexchanging the crystallization mother liquor with an artificial motherliquor.
 14. The crystallization method according to any one of claims1-5, wherein the batch volume is in the range of about 1 ml to about20,000 liters.
 15. A crystal of an anti-human IL-12 antibody.
 16. Acrystal of an anti-human IL-12 antibody, obtainable by a crystallizationmethod according to any one of claims 1-5.
 17. The crystal according toclaim 15, wherein the crystal has a sword-like morphology.
 18. Thecrystal according to claim 15, wherein the antibody is a polyclonalantibody or a monoclonal antibody.
 19. The crystal according to claim18, wherein the antibody is selected from the group consisting of achimeric antibody, a humanized antibody, a non-glycosylated antibody, ahuman antibody, and a mouse antibody.
 20. The crystal according to claim18, wherein the antibody is an IgG antibody.
 21. The crystal accordingto claim 20, wherein the antibody is selected from the group consistingof an IgG1, an IgG2, an IgG3 and an IgG4 antibody.
 22. The crystalaccording to claim 21, wherein the antibody is an anti-human IL-12antibody of the group IgG1.
 23. The crystal according to claim 22,wherein the crystal is prepared from an isolated human antibody thatdissociates from human IL-12 with a Kd of 1×10⁻¹⁰ M or less and ak_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance.
 24. The crystal according to claim 22, wherein thecrystal is prepared from an isolated human antibody with a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO:
 1. 25. The crystal according to claim 15, whereinthe antibody is ABT-874.
 26. A pharmaceutical composition comprising:(a) crystals of an anti-human IL-12 antibody according to claim 15, and(b) at least one pharmaceutical excipient; wherein the composition isprovided as a solid, a semisolid or a liquid formulation, eachformulation containing the antibody in crystalline form.
 27. Apharmaceutical composition comprising: (a) crystals of an anti-humanIL-12 antibody according to claim 15, and (b) at least onepharmaceutical excipient, which embeds or encapsulates the crystals. 28.The composition of claim 26 or 27, wherein the composition has anantibody concentration greater than about 1 mg/ml.
 29. The compositionof claim 28, wherein the composition has an antibody concentrationgreater than about 200 mg/ml.
 30. The composition according to claim 26and 27, wherein the composition comprises at least one carrier selectedfrom the group consisting of a polymeric biodegradable carrier, apolymeric non-biodegradable carrier, an oil carrier, and a lipidcarrier.
 31. The composition according to claim 30, wherein thepolymeric carrier is a polymer selected from one or more of the groupconsisting of: poly (acrylic acid), poly (cyanoacrylates), poly (aminoacids), poly (anhydrides), poly (depsipeptide), poly (esters), poly(lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly(ethylene glycol), poly (hydroxypropyl) methacrylamide, poly[(organo)phosphazene, poly (ortho esters), poly (vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends andcopolymers thereof.
 32. An injectable liquid composition comprisinganti-human IL-12 antibody crystals according to claim 15 and having anantibody concentration in the range of about 10 to about 400 mg/ml. 33.A crystal slurry composition comprising anti-human IL12 antibodycrystals according to claim 15, having an antibody concentration greaterthan about 100 mg/ml.
 34. A method for treating a mammal comprising thestep of administering to the mammal an effective amount of anti-humanIL-12 antibody crystals according to claim
 15. 35. A method for treatinga mammal comprising the step of administering to the mammal an effectiveamount of the composition according to claims 26 or
 27. 36. The methodaccording to claim 35, wherein the composition is administered by aparenteral route, an oral route, or by an injection.
 37. A method oftreating an IL-12-related disorder in a subject, which method comprisesadministering a therapeutically effective amount of the antibodycrystals according to claim
 15. 38. The method of claim 37, wherein theIL-12-related disorder is selected from the group consisting ofrheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lymearthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,systemic lupus erythematosus, Crohn's disease, ulcerative colitis,inflammatory bowel disease, insulin dependent diabetes mellitus,thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, atopic dermatitis, graft versus host disease, organtransplant rejection, acute or chronic immune disease associated withorgan transplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadic,polyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia areata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis C, common variedimmunodeficiency (common variable hypogammaglobulinaemia), dilatedcardiomyopathy, female infertility, ovarian failure, premature ovarianfailure, fibrotic lung disease, cryptogenic fibrosing alveolitis,post-inflammatory interstitial lung disease, interstitial pneumonitis,connective tissue disease associated interstitial lung disease, mixedconnective tissue disease associated lung disease, systemic sclerosisassociated interstitial lung disease, rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus associated lungdisease, dermatomyositis/polymyositis associated lung disease,Sjodgren's disease associated lung disease, ankylosing spondylitisassociated lung disease, vasculitic diffuse lung disease, haemosiderosisassociated lung disease, drug-induced interstitial lung disease,radiation fibrosis, bronchiolitis obliterans, chronic eosinophilicpneumonia, lymphocytic infiltrative lung disease, postinfectiousinterstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediatedhypoglycemia, type B insulin resistance with acanthosis nigricans,hypoparathyroidism, acute immune disease associated with organtransplantation, chronic immune disease associated with organtransplantation, osteoarthrosis, primary sclerosing cholangitis,idiopathic leucopenia, autoimmune neutropenia, renal disease NOS,glomerulonephritides, microscopic vasulitis of the kidneys, lymedisease, discoid lupus erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes),insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia,idiopathic thrombocytopenia, autoimmune thyroid disease,hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto'sdisease), atrophic autoimmune hypothyroidism, primary myxoedema,phacogenic uveitis, primary vasculitis and vitiligo. The humanantibodies, and antibody portions of the invention can be used to treatautoimmune diseases, in particular those associated with inflammation,including, rheumatoid spondylitis, allergy, autoimmune diabetes,autoimmune uveitis.
 39. The use of anti-human IL-12 antibody crystalsaccording to claim 15 for preparing a pharmaceutical composition fortreating an IL-12-related disease.
 40. Anti-human IL-12 antibodycrystals according to claim 15 for use in medicine.
 41. Thecrystallization method according to any one of claims 1-5, furthercomprising the step of extending the yield of the crystals by addingadditional polyalkylene glycol.
 42. The method according to claim 41,wherein the polyalkylene glycol is polyethylene glycol.
 43. The methodaccording to claim 41, wherein the polyalkylene glycol is addedcontinuously.
 44. The crystallization method according to according toclaim 1-5, further comprising the step of seeding the reaction withABT-874.